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Extracellular vesicles from adipose tissue-derived stem cells alleviate osteoporosis through osteoprotegerin and miR-21-5p.

ABSTRACT: Osteoporosis is one of the most common skeletal disorders caused by the imbalance between bone formation and resorption, resulting in quantitative loss of bone tissue. Since stem cell-derived extracellular vesicles (EVs) are growing attention as novel cell-free therapeutics that have advantages over parental stem cells, the therapeutic effects of EVs from adipose tissue-derived stem cells (ASC-EVs) on osteoporosis pathogenesis were investigated. ASC-EVs were isolated by a multi-filtration system based on the tangential flow filtration (TFF) system and characterized using transmission electron microscopy, dynamic light scattering, zeta potential, flow cytometry, cytokine arrays, and enzyme-linked immunosorbent assay. EVs are rich in growth factors and cytokines related to bone metabolism and mesenchymal stem cell (MSC) migration. In particular, osteoprotegerin (OPG), a natural inhibitor of receptor activator of nuclear factor-κB ligand (RANKL), was highly enriched in ASC-EVs. We found that the intravenous administration of ASC-EVs attenuated bone loss in osteoporosis mice. Also, ASC-EVs significantly inhibited osteoclast differentiation of macrophages and promoted the migration of bone marrow-derived MSCs (BM-MSCs). However, OPG-depleted ASC-EVs did not show anti-osteoclastogenesis effects, demonstrating that OPG is critical for the therapeutic effects of ASC-EVs. Additionally, small RNA sequencing data were analysed to identify miRNA candidates related to anti-osteoporosis effects. miR-21-5p in ASC-EVs inhibited osteoclast differentiation through Acvr2a down-regulation. Also, let-7b-5p in ASC-EVs significantly reduced the expression of genes related to osteoclastogenesis. Finally, ASC-EVs reached the bone tissue after they were injected intravenously, and they remained longer. OPG, miR-21-5p, and let-7b-5p in ASC-EVs inhibit osteoclast differentiation and reduce gene expression related to bone resorption, suggesting that ASC-EVs are highly promising as cell-free therapeutic agents for osteoporosis treatment.

SUBMITTER: Lee KS

PROVIDER: S-EPMC8485335 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionIt is essential to acknowledge that the cardiovascular toxicity associated with anthracycline drugs can be partially attributed to the damage inflicted on blood vessels and endothelial cells. Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have the potential to repair cellular processes and promote tissue regeneration through the transfer of signaling molecules such as miRNAs. In the present study, we investigated the effects of MSC-EVs on daunorubicin (DNR)-damaged human cardiac microvascular endothelial cells (HCMEC) and developing blood vessels of Chicken Chorioallantoic Membrane (CAM) in vivo.Materials and methodsWe constructed in vitro and in vivo models of DNR-damaged endothelial cells and developing blood vessel. Scratch wound assays, EdU assays, tube formation assays, and SA-β-Gal staining were used to evaluate the effects of MSC-EVs on cell migration, proliferation, angiogenesis capacity and cell senescence. Blood vessel area was used to assess the effects of MSC-EVs on CAM vasculature. RT-qPCR was used to detect the mRNA expression levels of inflammatory molecules. RNA sequencing was employed to compare differential gene expression and downstream regulatory mechanisms. RNA interference experiments and miRNA mimic overexpression experiments were used to validate the regulatory effects of target genes and downstream signaling pathways.ResultsWe found that MSC-EVs improved the migration, proliferation, and angiogenesis of HCMEC, while also alleviating cellular senescence. The angiogenic effect on the developing blood vessels was confirmed in vivo. We identified that MSC-EVs downregulated the expression of PARP9, thereby inhibiting the STAT1/pSTAT1 signaling pathway. This downregulation effect is likely mediated by the transfer of miR-186-5p from MSC-EVs to HCMEC. Overexpression of miR-186-5p in DNR-damaged HCMEC also exhibited the aforementioned downregulation effect. In vivo, the introduction of miR-186-5p mimics enhanced angiogenesis in the CAM model.ConclusionsTo summarize, our study reveals that MSC-EVs can restore the cellular function of DNR-damaged HCMEC and alleviate cellular senescence through the miR-185-5p-PARP9-STAT1/pSTAT1 pathway. This finding highlights the potential of MSC-EVs as a therapeutic strategy for mitigating the detrimental effects of anthracycline-induced endothelial damage.

Project description:BackgroundMesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have been found to have a great potential for soft tissue repair due to various biological functions, including pro-angiogenesis and low immunogenicity. However, the low yield and heterogeneity of MSC-EVs limited their clinical transformation. This study was designed to develop a novel adipose-derived stem cell engineered nanovesicles (ADSC-NVs) with high production and explore its pro-angiogenetic effect and application in adipose tissue regeneration.MethodsAdipose-derived stem cell-derived extracellular vesicles (ADSC-EVs) were isolated from an EVs-free culture medium for human ADSCs (hADSCs). ADSC-NVs were prepared by sequentially extruding ADSCs followed by iodixanol density gradient ultracentrifugation and were compared with ADSC-EVs in morphology, size distribution, protein contents and yield. The pro-angiogenetic effect of ADSC-NVs in different doses (0, 5, 20 and 80 μg/mL) in vitro was determined using transwell assay, tube formation assay, western blot and qRT-PCR. In vivo, BALB/c nude mice were administered injection of a mixture of fat granules and different dose of ADSC-NVs and grafts were harvested at 12 weeks post-transplantation for further analysis. By analyzing the weight and volume of grafts and histological evaluation, we investigated the effect of ADSC-NVs in vessel formation and adipose tissue regeneration.ResultsOur results showed yield of purified ADSC-NVs was approximately 20 times more than that of ADSC-EVs secreted by the same number of ADSCs. In vitro, both ADSC-NVs and ADSC-EVs exhibited a dose-dependent pro-angiogenetic effect, despite their distinct miRNA profiles. These effects of ADSC-NVs may be mediated by enriched miR-21-5p via PTEN inhibition and PI3K/p-Akt signaling activation. Furthermore, after a mixed injection of ADSC-NVs, vessel formation and adipose regeneration were observed in vivo in fat implants.ConclusionsOur study developed a potent alternative of ADSC-EVs. ADSC-NVs have a high pro-angiogenesis potential and can be used as cell-free therapeutic biomaterials in soft tissue regeneration.

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Extracellular vesicles from adipose tissue-derived stem cells alleviate osteoporosis through osteoprotegerin and miR-21-5p.

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