Logic-Based Strategy for Spatiotemporal Release of Dual Extracellular Vesicles in Osteoarthritis Treatment.

To effectively treat osteoarthritis (OA), the existing inflammation must be reduced before the cartilage damage can be repaired; this cannot be achieved with a single type of extracellular vesicles (EVs). Here, a hydrogel complex with logic-gates function is proposed that can spatiotemporally controlled release two types of EVs: interleukin 10 (IL-10)+ EVs to promote M2 polarization of macrophage, and SRY-box transcription factor 9 (SOX9)+ EVs to increase cartilage matrix synthesis. Following dose-of-action screening, the dual EVs are loaded into a matrix metalloporoteinase 13 (MMP13)-sensitive self-assembled peptide hydrogel (KM13E) and polyethylene glycol diacrylate/gelatin methacryloyl-hydrogel microspheres (PGE), respectively. These materials are mixed to form a "microspheres-in-gel" KM13E@PGE system. In vitro, KM13E@PGE abruptly released IL-10+ EVs after 3 days and slowly released SOX9+ EVs for more than 30 days. In vivo, KM13E@PGE increased the CD206+ M2 macrophage proportion in the synovial tissue and decreased the tumor necrosis factor-α and IL-1ß levels. The aggrecan and SOX9 expressions in the cartilage tissues are significantly elevated following inflammation subsidence. This performance is not achieved using anti-inflammatory or cartilage repair therapy alone. The present study provides an injectable, integrated delivery system with spatiotemporal control release of dual EVs, and may inspire logic-gates strategies for OA treatment.

SIRT3 and SIRT4 double-genes remodeled the mitochondrial network to induce hepatocellular carcinoma cell line differentiation and suppress malignant phenotypes.

Tumor cells with damaged mitochondria undergo metabolic reprogramming, but gene therapy targeting mitochondria has not been comprehensively reported. In this study, plasmids targeting the normal hepatocyte cell line (L-O2) and hepatocellular carcinoma cell line were generated using three genes SIRT3, SIRT4, and SIRT5. These deacetylases play a variety of regulatory roles in cancer and are related to mitochondrial function. Compared with L-O2, SIRT3 and SIRT4 significantly ameliorated mitochondrial damage in HCCLM3, Hep3B and HepG2 cell lines and regulated mitochondrial biogenesis and mitophagy, respectively. We constructed double-gene plasmid for co-express SIRT3 and SIRT4 using the internal ribosome entry site (IRES). The results indicated that the double-gene plasmid effectively expressed SIRT3 and SIRT4, significantly improved mitochondrial quality and function, and reduced mtDNA level and oxidative stress in HCC cells. MitoTracker analysis revealed that the mitochondrial network was restored. The proliferation, migration capabilities of HCC cells were reduced, whereas their differentiation abilities were enhanced. This study demonstrated that the use of IRES-linked SIRT3 and SIRT4 double-gene vectors induced the differentiation of HCC cells and inhibited their development by ameliorating mitochondrial dysfunction. This intervention helped reverse metabolic reprogramming, and may provide a groundbreaking new framework for HCC treatment.

Injectable MMP1-sensitive microspheres with spatiotemporally controlled exosome release promote neovascularized bone healing.

Bone marrow mesenchymal stromal cell-derived exosomes (BMSC-Exos) can recruit stem cells for bone repair, with neovessels serving as the main migratory channel for stem cells to the injury site. However, existing exosome (Exo) delivery strategies cannot reach the angiogenesis phase following bone injury. To that end, an enzyme-sensitive Exo delivery material that responds to neovessel formation during the angiogenesis phase was designed in the present study to achieve spatiotemporally controlled Exo release. Herein, matrix metalloproteinase-1 (MMP1) was found to be highly expressed in neovascularized bone; as a result, we proposed an injectable MMP1-sensitive hydrogel microspheres (KGE) made using a microfluidic chip prepared by mixing self-assembling peptide (KLDL-MMP1), GelMA, and BMSC-Exos. The results revealed that KGE microspheres had a uniform diameter of 50-70 µm, ideal for minimally invasive injection and could release exosomes in response to MMP1 expression. In vitro experiments demonstrated that KGE had less cytotoxicity and could promote the migration and osteodifferentiation of BMSCs. Furthermore, in vivo experiments confirmed that KGE could promote bone repair during angiogenesis by recruiting CD90+ stem cells via neovessels. Collectively, our results suggest that injectable enzyme-responsive KGE microspheres could be a promising Exo-secreting material for accelerating neovascularized bone healing. STATEMENT OF SIGNIFICANCE: Exosomes can spread through blood vessels and activate stem cells to participate in bone repair, but under normal circumstances, exosomes lacking sustained-release delivery materials cannot be maintained until the angiogenesis phase. In this study, we found that MMP1 was highly expressed in neovascularized bone, then we proposed an MMP1-sensitive injectable microsphere that carries exosomes and responds temporally and spatially to neovascularization, which maximizes the ability of exosomes to recruit stem cells. Different from previous strategies that focus on promoting angiogenesis to accelerate bone healing, this is a brand new delivery strategy that is stimuli-responsive to neovessel formation. In addition, the preparation of self-assembled peptide microspheres by a microfluidic chip is also proposed for the first time.

Herpes Simplex Type 1 UL43 Multiple Membrane-Spanning Protein Increases Energy Metabolism in Host Cells through Interacting with ARL2.

Non-essential proteins for viral replication affect host cell metabolism, while the function of the UL43 protein of herpes simplex virus 1 (HSV-1) is not clear. Herein, we performed a comprehensive microarray analysis of HUVEC cells infected with HSV-1 and its UL43-deficient mutant and found significant variation in genes associated with cellular energy metabolic pathways. The localization of UL43 protein in host cells and how it affects cellular energy metabolism pathways were further investigated. Internalization analysis showed that the UL43 protein could be endocytosis-mediated by YPLF motif (aa144-147) and localized to mitochondria. At the same time, more ATP was produced by coupling with mitochondrial small G protein ARF-like 2 (ARL2) GTPase, which triggered the phosphorylation of ANT1 (SLC25A4) to affect the opening degree of mitochondrial permeability transition pore (mPTP), and significantly promoted the aerobic oxidation and oxidative phosphorylation of glucose. Our study shows that UL43 mediates the improvement of host cell metabolism after HSV-1 infection. Additionally, UL43 protein could be a valuable ATP-stimulating factor for mammalian cells.

Gallic Acid Ameliorates the Inflammatory State of Periodontal Ligament Stem Cells and Promotes Pro-Osteodifferentiation Capabilities of Inflammatory Stem Cell-Derived Exosomes.

The slow proliferation rate and poor osteodifferentiation ability of inflammatory periodontal membrane stem cells extracted from periodontitis tissues (i-PDLSCs) account for poor efficiency in treating inflammatory bone loss. Exosomes reportedly have inducible and relatively stable components, allowing them to promote inflammatory bone repair, but obtaining i-PDLSCs exosomes with the ability to promote osteodifferentiation is challenging. In the present study, i-PDLSCs were extracted from periodontal membrane tissues of patients with severe periodontitis, and in vitro induction with gallic acid (GA) significantly promoted the proliferative activity of i-PDLSCs at a concentration of 10 mM, with TC0 of 11.057 mM and TC50 of 67.56 mM for i-PDLSCs. After mRNA sequencing, we found that GA could alleviate oxidative stress in i-PDLSCs and increase its mitochondrial membrane potential and glucose aerobic metabolism level, thus promoting the osteodifferentiation of i-PDLSCs. After exosomes of i-PDLSCs after GA induction (i-EXO-GA) were isolated by differential centrifugation, we found that 200 ug/mL of i-EXO-GA could remarkably promote the osteodifferentiation of i-PDLSCs. Overall, our results suggest that GA induction can enhance the proliferation and osteodifferentiation in primary cultures of i-PDLSCs in vitro, mediated by alleviating oxidative stress and glycometabolism levels in cells, which further influences the osteodifferentiation-promoting ability of i-EXO-GA. Overall, we provide a viable cell and exosome induction culture method for treating inflammatory alveolar defects associated with periodontitis.

Receptor activator of nuclear factor-κB ligand and tumor necrosis factor-α promotes osteoclast differentiation through the exosomes of inflammatory periodontal ligament stem cells.

OBJECTIVES: Pathological bone resorption is common in chronic periodontitis. However, the effect of exosomes (Exo) secreted by periodontal ligament stem cells (PDLSCs) on bone resorption is unclear. This study explored the Exo of inflammatory PDLSCs, their protein components, and their effects on osteoclast differentiation. METHODS: PDLSCs were isolated from the periodontal ligament tissues of orthodontic patients and those with chronic periodontitis. The surface markers of PDLSCs were detected by flow cytometry. Exo were characterized by Western blot, transmission electron microscope (TEM), bicinchoninic acid assay (BCA), nanosight tracking analysis (NTA). The protein components of Exo were detected by protein profiling. The expression levels of differentially expressed proteins tumor necrosis factor-α (TNF-α), receptor activator of nuclear factor-κB ligand (RANKL), interleukin (IL)-1α, transforming growth factor ß (TGF-ß), and bone morphogenetic protein 2 (BMP-2) were verified by enzyme linked immunosorbent assay (ELISA). Then, 10, 100, and 1 000 µg·mL-1 of Exo-CP or Exo-WT were added to RAW264.7 medium, and the expression levels of osteoclast-related indicators were detected by real time quantitative polymerase chain reaction (RT-qPCR), Western blot, and tartrate resistant acid phosphatase (TRAP) staining at 5 days. Experimental data were statistically analyzed using SPSS 24.0 software. RESULTS: The differentially expressed proteins enriched in Exo-CP were mainly related to the tumor necrosis factor (TNF) signaling, osteoclast differentiation, and nuclear transcription factor κB (NF-κB) signaling pathways. ELISA experiments confirmed Exo-CP had high expression of TNF-α, RANKL, and IL-1α and low expression of TGF-ß1 and BMP-2 (P<0.05). Adding Exo-CP to RAW264.7 significantly increased the expression of mRNA and proteins related to osteoclast differentiation of cells. In a concentration-dependent manner, the effect of Exo-CP on osteoclast differentiation at concentrations of 100 and 1 000 µg·mL-1 was significantly higher than that on the 10 µg·mL-1 concentration group (P<0.05). CONCLUSIONS: Pathological bone resorption of chronic periodontitis may be caused by the activation of Exo-CP to promote osteoclast differentiation. The main protein in Exo may be RANKL and TNF-α. This research provides a new perspective on pathological bone resorption in chronic periodontitis.

The UL16 protein of HSV-1 promotes the metabolism of cell mitochondria by binding to ANT2 protein.

Long-term studies have shown that virus infection affects the energy metabolism of host cells, which mainly affects the function of mitochondria and leads to the hydrolysis of ATP in host cells, but it is not clear how virus infection participates in mitochondrial energy metabolism in host cells. In our study, HUVEC cells were infected with HSV-1, and the differentially expressed genes were obtained by microarray analysis and data analysis. The viral gene encoding protein UL16 was identified to interact with host protein ANT2 by immunoprecipitation and mass spectrometry. We also reported that UL16 transfection promoted oxidative phosphorylation of glucose and significantly increased intracellular ATP content. Furthermore, UL16 was transfected into the HUVEC cell model with mitochondrial dysfunction induced by D-Gal, and it was found that UL16 could restore the mitochondrial function of cells. It was first discovered that viral protein UL16 could enhance mitochondrial function in mammalian cells by promoting mitochondrial metabolism. This study provides a theoretical basis for the prevention and treatment of mitochondrial dysfunction or the pathological process related to mitochondrial dysfunction.