Background: Bone tissue engineering offers a new approach for the treatment of bone defects, with angiogenesis being critical to the survival and development of tissue-engineered bone. Mineralized osteoblasts (MOBs) have been reported to promote vascular formation by endothelial cells (ECs) through the secretion of exosomes containing a variety of angiogenic factors. The aim of the present study was to investigate the effect of miR-423-5p contained within exosomes derived from MOBs (MOB-Exos) on EC angiogenesis. Methods: The Cell Counting Kit-8 (CCK-8), scratch wound healing, Transwell migration, and tube formation assays were conducted to assess the in vitro effects of MOB-Exos on EC proliferation, migration, and tubule-forming capabilities. The miR-423-5p level in MOB-Exos was quantified using quantitative polymerase chain reaction (qPCR). Co-culture experiments were used to study the exosomal transport of miR-423-5p and its angiogenic effects. High-throughput sequencing was used to identify differentially expressed genes, and a dual luciferase reporter assay to determine whether CXCL10 was a direct target gene for miR-423-5p. Furthermore, the in vivo effect of MOB-Exos-derived miR-423-5p on angiogenesis was evaluated using a subcutaneous xenograft model. Results: MOB-Exos significantly promoted the in vitro proliferation, migration, and tubule formation of ECs. A high level of miR-423-5p was found in MOB-Exos and promoted the angiogenesis of ECs. The CXCL10 gene was significantly downregulated in ECs upon miR-423-5p mimic transfection. Dual luciferase reporter assay confirmed the direct binding of miR-423-5p to the CXCL10 gene. miR-423-5p derived from MOB-Exos upregulated expression of the vascular markers CD31 and vascular endothelial growth factor (VEGF) in vivo, thus underscoring its angiogenic potential. Conclusion: This study found that miR-423-5p derived from MOB-Exos could potentially enhance EC angiogenesis via the regulation of CXCL10. Therefore, exosomes are promising therapeutic candidates for clinical bone defects.
Exosomes secreted from osteoblasts (OBs) can regulate the angiogenesis of endothelial cells (ECs); however, whether cerebrospinal fluid pulsation (CSFP) stress, a special mechanical stimulation, can influence the cell’s communication in the context of angiogenesis remains unknown. In this study, the effect of exosomes derived from CSFP stress-stimulated OBs on facilitating the angiogenesis of ECs was investigated. First, OBs were cultured in a CSFP bioreactor, and exosomes derived from OBs were isolated and identified. Cell Counting Kit 8 assay, transwell migration assay, wound healing migration assay, and tube formation assay were conducted to assess the effects of CSFP stress-stimulated OBs-derived exosomes (CSFP-Exos) on the angiogenesis of ECs. Then high-throughput RNA sequencing was used to determine the miRNA profiles of Non-CSFP stress-stimulated OBs-derived exosomes (NCSFP-Exos) and CSFP-Exos, and the luciferase reporter gene assay was performed to confirm the binging of miR-423-5p to DUSP8. In addition, the Matrigel plug assay was performed to explore whether exosomal miR-423-5p has the same effects in vivo . Our results suggested that CSFP-Exos can promote the angiogenesis of ECs, and miR-423-5p was enriched in CSFP-Exos. Moreover, miR-423-5p could promote the effect of angiogenesis via directly targeting dual-specificity phosphatase 8 (DUSP8), which inhibited the ERK1/2 signaling pathway. In conclusion, exosomal miR-423-5p derived from CSFP stress-stimulated OBs could promote the angiogenesis of ECs by the DUSP8/ERK1/2 signaling pathway.
Liu Hong,Zhang Shuang,Zhao Xiu‑Nan,Wang Zhen‑Zhen,Song Jia‑Ni,Liu Shu‑Xin
Purpose Membranous nephropathy (MN) is the most common pathological type of primary nephrotic syndrome in adults. MicroRNAs (miRNAs) are involved in the pathogenesis of several renal diseases. Here we investigated the potential involvement of miRNAs in MN progression. Methods miRNA sequencing was performed in the urinary exosomes of MN patients and normal controls (three cases per group). An experimental mouse MN model induced by cationic bovine serum albumin (CBSA) and MPC5 podocytes intoxicated by puromycin aminonucleoside (PAN) were used in this study. Results Compared with the normal controls, 19 miRNAs were downregulated and 13 upregulated in the urinary exosomes of MN patients. In an expanded cohort, we confirmed that miR-423-5p was significantly upregulated in the urinary exosomes of MN patients. Urinary particle concentrations and size distribution were assessed using transmission electron microscopy and NanoSight. Similarly, upregulated miR-423-5p was observed in PAN-intoxicated MPC5 cells. Lentiviral vector-mediated inhibition of miR-423-5p suppressed podocyte apoptosis and inactivated β-catenin signaling. Upon miRNA database prediction, WT1 was identified as a target of miR-423-5p. Consistently, downregulated WT1 expression in PAN-intoxicated MPC5 cells was rescued by miR-423-5p inhibition. The target relationship between miR-423-5p and WT1 mRNA 3’UTR was validated by the dual luciferase reporter assay. Knockdown of WT1 reversed the inhibitory effect of LV-anti-miR-423-5p on podocyte apoptosis and β-catenin signaling. Notably, miR-423-5p inhibition and WT1 overexpression attenuated renal injury and podocyte apoptosis in MN mice. Conclusion Our study demonstrates that miR-423-5p expression is upregulated in urinary exosomes of MN patients and modulates WT1, affecting podocyte apoptosis by the β-catenin signaling pathway. Inhibition of miR-423-5p action may protect against MN.
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