Background: Blood contains exosomes that are related to tumor cells. Those exosomes can regulate communication between cells and have a great influence on a variety of tumor-associated proceedings through their target cells. Therefore, serum exosomes (SE) were supposed to play a crucial role in cancer development. Methods: This study presented a quantitative proteomics analysis to identify the protein content in SEs including 30 subjects from oral squamous cell carcinoma (OSCC) patients with lymph node metastasis (LNM), OSCC patients with no LNM (NLNM), and healthy controls (HC). Differentially expressed proteins (DEP) were analyzed by bioinformatics, and then a total of 30 subjects were used for Western blot and 60 subjects for IHC, ELISA, and RT-PCR verifications. The correlations were assessed between DEP expression and clinicopathologic factors. Results: A total of 415 proteins were identified. Comparing with HC and OSCC-NLNM, we found 37 proteins and 28 proteins in the SEs of OSCC-LNM, respectively. There were significant correlations among the expression of PF4V1 with tumor differentiation level, PF4V1 and F13A1 with the number of positive nodes, and ApoA1 with smoking and drinking. ROC curve analysis indicated that the combinations of the different biomarkers or specimen were obviously superior to single biomarker or specimen for diagnosing OSCC-LNM. Conclusions: We conclude that PF4V1, CXCL7, F13A1, and ApoA1 from SEs may be related to the metastasis of OSCC, which would be helpful in the diagnosis of OSCC-LNM. Impact: Biomarkers from SEs could help with the diagnosis of metastasis in OSCC.

Distraction osteogenesis (DO) is a widely used bone regenerative technique. However, the DO process is slow, and the consolidation phase is long. Therefore, it is of great clinical significance to explore the mechanism of DO, and shorten its duration. Recent studies reported that stem cell exosomes may play an important role in promoting angiogenesis related to DO, but the mechanism remains unclear. Canine endothelial colony-forming cells (ECFCs) were isolated and cultured, and the expression of THBS1 in canine ECFCs were inhibited using a lentiviral vector. The exosomes secreted by canine ECFCs were isolated and extracted, and the effect of exosomes on the angiogenic activity of Human umbilical vein endothelial cells (HUVECs) was detected by proliferation, migration, and tube formation experiments. WB and qRT-PCR were used to explore the effects and mechanisms of THBS1-mediated ECFC-Exos on HUVECs angiogenesis. Then, a mandibular distraction osteogenesis (MDO) model was established in adult male beagles, and exosomes were injected into the canine peripheral blood. Micro-CT, H&E, Masson, and IHC staining were used to explore the effects and mechanisms of THBS1-mediated ECFC-Exos on angiogenesis and osteogenesis in the DO area. ECFC-Exo accelerated HUVECs proliferation, migration and tube formation, and this ability was enhanced by inhibiting the expression of THBS1 in ECFC-Exo. Using Western blot-mediated detection, we demonstrated that inhibiting THBS1 expression in ECFCs-Exo activated PI3K, AKT, and ERK phosphorylation levels in HUVECs, which promoted VEGF and bFGF expressions. In the DO model of the canine mandible, ECFCs-Exo injected into the peripheral blood aggregated into the DO gap, thus promoting angiogenesis and bone formation in the DO tissue by reducing THBS1 expression in ECFC-Exo. Our findings suggested that ECFC-Exos markedly enhances angiogenesis of endothelial cells, and promotes bone healing in canine MDO. Thus, THBS1 plays a crucial role in the ECFC-Exos-mediated regulation of canine MDO angiogenesis and bone remodeling. This study reveals that the angiogenic promotion via THBS1 suppression in ECFC-Exos may be a promising strategy for shortening the DO duration.

Distraction osteogenesis (DO) is used to treat large bone defects in the field of oral and maxillofacial surgery. Successful DO-mediated bone regeneration is dependent upon angiogenesis, and endothelial progenitor cells (EPCs) are key mediators of angiogenic processes. The N6-methyladenosine (m⁶A) methyltransferase has been identified as an important regulator of diverse biological processes, but its role in EPC-mediated angiogenesis during DO remains to be clarified. In the present study, we found that the level of m⁶A modification was significantly elevated during the process of DO and that it was also increased in the context of EPC angiogenesis under hypoxic conditions, which was characterized by increased METTL3 levels. After knocking down METTL3 in EPCs, m⁶A RNA methylation, proliferation, tube formation, migration, and chicken embryo chorioallantoic membrane (CAM) angiogenic activity were inhibited, whereas the opposite was observed upon the overexpression of METTL3. Mechanistically, METTL3 silencing reduced the levels of VEGF and PI3Kp110 as well as the phosphorylation of AKT, whereas METTL3 overexpression reduced these levels. SC79-mediated AKT phosphorylation was also able to restore the angiogenic capabilities of METTL3-deficient EPCs in vitro and ex vivo. In vivo, METTL3-overexpressing EPCs were additionally transplanted into the DO callus, significantly enhancing bone regeneration as evidenced by improved radiological and histological manifestations in a canine mandibular DO model after consolidation over a 4-week period. Overall, these results indicate that METTL3 accelerates bone regeneration during DO by enhancing EPC angiogenesis via the PI3K/AKT pathway.

Background: Distraction osteogenesis (DO) is an effective treatment in craniomaxillofacial surgery. However, the issue of sufficient blood supply at the regeneration tissue has limited its wide application. Panax notoginseng saponins (PNS) is a Traditional Chinese Medicine that is commonly used to treat a range of angiogenic diseases. However, the mechanisms whereby PNS alters angiogenesis in endothelial progenitor cells (EPCs) have yet to be clarified. Methods: EPCs were identified by immunofluorescence, confirmed by their uptake of fluorescently labeled Dil-ac-LDL and FITC-UEA-1. EPCs were treated with different concentrations of PNS, and the effects of PNS on cell proliferation were measured on the optimal concentration of PNS determined. The effects of PNS on angiogenesis and migration, angiogenic cytokines mRNA expression and the proteins of the Wnt pathway were investigated. Then knocked down β-catenin in EPCs and treated with the optimum concentrational PNS, their angiogenic potential was evaluated in tube formation and migration assays. In addition, the expression of cytokines associated with angiogenesis and Wnt/β-catenin was then assessed via WB and RT-qPCR. Results: We were able to determine the optimal concentration of PNS in the promotion of cell proliferation, tube formation, and migration to be 6.25 mg/L. PNS treatment increased the mRNA levels of VEGF, bFGF, VE-Cadherin, WNT3a, LRP5, β-catenin, and TCF4. After knocked down β-catenin expression, we found that PNS could sufficient to partially reverse the suppression of EPC angiogenesis. Conclusions: Overall, 6.25 mg/L PNS can promote EPC angiogenesis via Wnt/β-catenin signaling pathway activation.

In addition to its important transport functions, the skeletal system is involved in complex biological activities for the regulation of blood vessels. Endothelial progenitor cells (EPCs), as stem cells of endothelial cells (ECs), possess an effective proliferative capacity and a powerful angiogenic capacity prior to their differentiation. They demonstrate synergistic effects to promote bone regeneration and vascularization more effectively by co-culturing with multiple cells. EPCs demonstrate a significant therapeutic potential for the treatment of various bone diseases by secreting a combination of growth factors, regulating cellular functions, and promoting bone regeneration. In this review, we retrospect the definition and properties of EPCs, their interaction with mesenchymal stem cells, ECs, smooth muscle cells, and immune cells in bone regeneration, vascularization, and immunity, summarizing their mechanism of action and contribution to bone biology. Additionally, we generalized their role and potential mechanisms in the treatment of various bone diseases, possibly indicating their clinical application.

Background: Distraction osteogenesis (DO) is a highly efficacious form of reconstructive bone regeneration, but its clinical utility is limited by the prolonged period required for bone consolidation to occur. Understanding the mechanistic basis for DO and shortening this consolidation phase thus represent promising approaches to improving the clinical utility of this procedure. Methods: A mandibular DO (MDO) canine model was established, after which small RNA sequencing was performed to identify relevant molecular targets genes. Putative miRNA target genes were identified through bioinformatics and confirmed through qPCR, Western blotting, and dual-luciferase reporter assays. Peripheral blood samples were collected to isolate serum and endothelial colony-forming cells (ECFCs) in order to measure miR-205, NOTCH2, and angiogenic cytokines expression levels. Lentiviral constructs were then used to inhibit or overexpress miR-205 and NOTCH2 in isolated ECFCs, after which the angiogenic activity of these cells was evaluated in migration, wound healing, proliferation, tube formation, and chick chorioallantoic membrane (CAM) assay. Autologous ECFCs transfected to knockdown miR-205 and were injected directly into the distraction callus. On days 14, 28, 35 and 42 after surgery, bone density was evaluated via CBCT, and callus samples were collected and evaluated via histological staining to analyze bone regeneration and remodeling. Results: MiR-205 was identified as being one of the miRNAs that was most significantly downregulated in MDO callus samples. Downregulation of miR-205 was also observed in DO-ECFCs and serum of animals undergoing MDO. Inhibiting miR-205 markedly enhanced angiogenesis, whereas overexpressing miR-205 had the opposite effect in vitro. Importantly, NOTCH2, which is a unique regulator in bone angiogenesis, was identified as a miR-205 target gene. Consistent with this regulatory relationship, knocking down NOTCH2 suppressed angiogenesis, and transduction with a miR-205 inhibitor lentivirus was sufficient to rescue angiogenic activity. When ECFCs in which miR-205 had been inhibited were transplanted into the MDO callus, this significantly bolstered osteogenesis, and remodeling in vivo. Conclusions: MiR-205 is a significant regulator of the MDO process, and inhibiting this miRNA can accelerate MDO-related mineralization. Overall, these results offer new insights into the mechanistic basis for this procedure, highlighting potential targets for therapeutic clinical intervention.