Engineering exosomes derived from subcutaneous fat MSCs specially promote cartilage repair as miR-199a-3p delivery vehicles in Osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease involving cartilage. Exosomes derived from Mesenchymal stem cells (MSCs) therapy improves articular cartilage repair, but subcutaneous fat (SC) stromal cells derived exosomes (MSCsSC-Exos), especially engineering MSCsSC-Exos for drug delivery have been rarely reported in OA therapy. This objective of this study was to clarify the underlying mechanism of MSCsSC-Exos on cartilage repair and therapy of engineering MSCsSC-Exos for drug delivery in OA. MSCsSC-Exos could ameliorate the pathological severity degree of cartilage via miR-199a-3p, a novel molecular highly enriched in MSCsSC-Exos, which could mediate the mTOR-autophagy pathway in OA rat model. Intra-articular injection of antagomiR-199a-3p dramatically attenuated the protective effect of MSCsSC-Exos-mediated on articular cartilage in vivo. Furthermore, to achieve the superior therapeutic effects of MSCsSC-Exos on injured cartilage, engineering exosomes derived from MSCsSC as the chondrocyte-targeting miR-199a-3p delivery vehicles were investigated in vitro and in vivo. The chondrocyte-binding peptide (CAP) binding MSCsSC-Exos could particularly deliver miR-199a-3p into the chondrocytes in vitro and into deep articular tissues in vivo, then exert the excellent protective effect on injured cartilage in DMM-induced OA mice. As it is feasible to obtain human subcutaneous fat from healthy donors by liposuction operation in clinic, meanwhile engineering MSCsSC-Exos to realize targeted delivery of miR-199a-3p into chondrocytes exerted excellent therapeutic effects in OA animal model in vivo. Through combining MSCsSC-Exos therapy and miRNA therapy via an engineering approach, we develop an efficient MSCsSC-Exos-based strategy for OA therapy and promote the application of targeted-MSCsSC-Exos for drug delivery in the future.

ADSCs increase the autophagy of chondrocytes through decreasing miR-7-5p in Osteoarthritis rats by targeting ATG4A.

BACKGROUND: Osteoarthritis (OA) is a highly degenerative joint disease, mainly companying with progressive destruction of articular cartilage. Adipose-derived stromal cells (ADSCs) therapy enhances articular cartilage repair, extracellular matrix (ECM) synthesis and attenuates joints inflammation, but specific mechanisms of therapeutic benefit remain poorly understood. This study aimed to clarify the therapeutic effects and mechanisms of ADSCs on cartilage damage in the keen joint of OA rat model. METHODS: Destabilization of the medial meniscus (DMM) and anterior cruciate ligament transection (ACLT) surgery-induced OA rats were treated with allogeneic ADSCs by intra-articular injections for 6 weeks. The protective effect of ADSCs in vivo was measured using Safranin O and fast green staining, immunofluorescence and western blot analysis. Meanwhile, the miRNA-7-5p (miR-7-5p) expression was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The mechanism of increased autophagy with ADSCs addition through decreasing miR-7-5p was revealed using oligonucleotides, and adenovirus in rat chondrocytes. The luciferase reporter assay revealed the molecular role of miR-7-5p and autophagy related 4A (ATG4A). The substrate of mTORC1 pathway: (p-)p70S6 and (p-)S6 in OA models with ADSCs addition were detected by western blotting. RESULTS: The ADSCs treatment repaired the articular cartilage and maintained chondrocytes ECM homeostasis through modulating chondrocytes autophagy in the OA model, indicators of the change of autophagic proteins expression and autophagic flux. Meanwhile, the increased autophagy induced by ADSCs treatment was closely related to the decreased expression of host-derived miR-7-5p, a negative modulator of OA progression. Functional genomics (overexpression of genes) in vitro studies demonstrate the inhibition of host-derived miR-7-5p in mediating the benefit of ADSCs administration in OA model. Then ATG4A was defined as a target gene of miR-7-5p, and the negative relation between miR-7-5p and ATG4A was investigated in the OA model treated with ADSCs. Furthermore, miR-7-5p mediated chondrocyte autophagy by targeting ATG4A in the OA model treated with ADSCs was confirmed with the rescue trial of ATG4A/miR-7-5p overexpression on rat chondrocyte. Finally, the mTORC1 signaling pathways mediated by host-derived miR-7-5p with ADSCs treatment were decreased in OA rats. CONCLUSIONS: ADSCs promote the chondrocytes autophagy by decreasing miR-7-5p in articular cartilage by targeting ATG4A and a potential role for ADSCs based therapeutics for preventing of articular cartilage destruction and extracellular matrix (ECM) degradation in OA.

ADSCs-derived exosomes ameliorate hepatic fibrosis by suppressing stellate cell activation and remodeling hepatocellular glutamine synthetase-mediated glutamine and ammonia homeostasis.

BACKGROUND: Hepatic fibrosis is a common pathologic stage in chronic liver disease development, which might ultimately lead to liver cirrhosis. Accumulating evidence suggests that adipose-derived stromal cells (ADSCs)-based therapies show excellent therapeutic potential in liver injury disease owing to its superior properties, including tissue repair ability and immunomodulation effect. However, cell-based therapy still limits to several problems, such as engraftment efficiency and immunoreaction, which impede the ADSCs-based therapeutics development. So, ADSCs-derived extracellular vesicles (EVs), especially for exosomes (ADSC-EXO), emerge as a promise cell-free therapeutics to ameliorate liver fibrosis. The effect and underlying mechanisms of ADSC-EXO in liver fibrosis remains blurred. METHODS: Hepatic fibrosis murine model was established by intraperitoneal sequential injecting the diethylnitrosamine (DEN) for two weeks and then carbon tetrachloride (CCl4) for six weeks. Subsequently, hepatic fibrosis mice were administrated with ADSC-EXO (10 µg/g) or PBS through tail vein infusion for three times in two weeks. To evaluate the anti-fibrotic capacity of ADSC-EXO, we detected liver morphology by histopathological examination, ECM deposition by serology test and Sirius Red staining, profibrogenic markers by qRT-PCR assay. LX-2 cells treated with TGF-ß (10 ng/ml) for 12 h were conducted for evaluating ADSC-EXO effect on activated hepatic stellate cells (HSCs). RNA-seq was performed for further analysis of the underlying regulatory mechanisms of ADSC-EXO in liver fibrosis. RESULTS: In this study, we obtained isolated ADSCs, collected and separated ADSCs-derived exosomes. We found that ADSC-EXO treatment could efficiently ameliorate DEN/CCl4-induced hepatic fibrosis by improving mice liver function and lessening hepatic ECM deposition. Moreover, ADSC-EXO intervention could reverse profibrogenic phenotypes both in vivo and in vitro, including HSCs activation depressed and profibrogenic markers inhibition. Additionally, RNA-seq analysis further determined that decreased glutamine synthetase (Glul) of perivenous hepatocytes in hepatic fibrosis mice could be dramatically up-regulated by ADSC-EXO treatment; meanwhile, glutamine and ammonia metabolism-associated key enzyme OAT was up-regulated and GLS2 was down-regulated by ADSC-EXO treatment in mice liver. In addition, glutamine synthetase inhibitor would erase ADSC-EXO therapeutic effect on hepatic fibrosis. CONCLUSIONS: These findings demonstrated that ADSC-derived exosomes could efficiently alleviate hepatic fibrosis by suppressing HSCs activation and remodeling glutamine and ammonia metabolism mediated by hepatocellular glutamine synthetase, which might be a novel and promising anti-fibrotic therapeutics for hepatic fibrosis disease.

The association between monocytic myeloid-derived suppressor cells levels and the anti-tumor efficacy of anti-PD-1 therapy in NSCLC patients.

Monocytic myeloid-derived suppressor cells (M-MDSCs), granulocytic MDSC (G-MDSCs) and regulatory T cells (Tregs) inhibit adaptive anti-tumor immunity and undermine the efficacy of anti-PD-1 therapy. However, the impact of anti-PD-1 treatment on these immunosuppressive cells has not been clearly defined in non-small cell lung cancer (NSCLC). In this retrospective study, 27 advanced NSCLC patients were divided into partial response (PR), stable disease (SD), and progressive disease (PD) groups. The impact of anti-PD-1 therapy on circulating Tregs, G-MDSCs, and M-MDSCs was assessed by flow cytometer. Here, we found that anti-PD-1 treatment boosted circulating Tregs levels, which presented the most remarkable augment during the first two therapeutic cycles, in NSCLC patients. In contrast, anti-PD-1 therapy did not overall change G-MDSCs and M-MDSCs levels. However, the PR group had a higher baseline level of M-MDSCs, which exhibited a significant decrease after the first cycle of anti-PD-1 treatment. Besides, M-MDSCs levels in the PR group were maintained at a low level in the following therapeutic cycles. Consistently, Tregs levels robustly increased in the syngeneic tumor model after anti-mouse PD-1 Ab treatment. Accordingly, M-MDSCs neutralization by anti-mouse ly6c Ab enhanced the anti-tumor efficacy of anti-PD-1 therapy in mice. Finally, the decreased M-MDSCs levels were associated with the enhanced effector CD8+ T cells expansion in the PR group and mice. In conclusion, anti-PD-1 therapy upregulates Tregs levels in NSCLC patients, and the M-MDSC levels are associated with the anti-tumor efficacy of anti-PD-1 treatment. Neutralization of M-MDSCs may be a promising option to augment anti-PD-1 therapy efficacy in NSCLC.

RUVBL1-ITFG1 interaction is required for collective invasion in breast cancer.

BACKGROUND: The mechanisms of breast cancer collective invasion are poorly understood limiting the metastasis therapy. The ATPase RUVBL1 is frequently overexpressed in various cancers and plays a crucial role in oncogenic process. We further investigated the role of RUVBL1 in promoting collective invasion and uncovered that targeting RUVBL1 could inhibit metastatic progression. METHODS: The expression levels of RUVBL1 and ITFG1 were examined by Western blot and qRT-PCR. Co-localization and interaction of RUVBL1 and ITFG1 were determined by immunofluorescence and co-immunoprecipitation. The invasive ability was examined by transwell assay and microfluidic assay. The metastatic and tumorigenic abilities of breast cancer cells were revealed in BALB/c nude mice by xenograft and tail vein injection. RESULTS: ATPase RUVBL1 is highly expressed in breast cancer and predicts the poor prognosis. Elevated expression of RUVBL1 is found in high metastatic breast cancer cells. Silencing RUVBL1 suppresses cancer cell expansion and invasion in vitro and in vivo. RUVBL1 interacts with a conserved transmembrane protein ITFG1 in cytoplasm and plasma membrane to promote the collective invasion. Using a microfluidic model, we demonstrated that silencing RUVBL1 or ITFG1 individually compromises collective invasion of breast cancer cells. CONCLUSION: RUVBL1 is a vital regulator for collective invasion. The interaction between RUVBL1 and ITFG1 is required for breast cancer cell collective invasion and progression. GENERAL SIGNIFICANCE: Targeting collective invasion promoted by RUVBL1-ITFG1 complex provides a novel therapeutic strategy to improve the prognosis of invasive breast cancer.