microRNA-106b derived from endothelial cell-secreted extracellular vesicles prevents skin wound healing by inhibiting JMJD3 and RIPK3.

Intriguingly, microRNAs (miRs) transferred as cargo in extracellular vesicles (EVs) can modulate wound healing through their regulation of fibroblast functions. In this study, we investigated the effects of miR-106b transfer via EVs derived from human umbilical vein endothelial cells (HUVECs) on skin wound healing. Dual-luciferase reporter gene assay identified that miR-106b could target and inhibit JMJD3. RT-qPCR analysis showed EVs isolated from HUVECs had enriched expression of miR-106b. LL29 fibroblast cells and HaCaT keratinocytes were co-cultured with HUVEC-derived EVs, in which miR-106b had been up-regulated or down-regulated by its mimic or inhibitor. The co-culture with HUVEC-derived EVs increased miR-106b expression, and reduced the viability and adhesion of LL29 and HaCaT cells, whereas the inhibition of miR-106b in HUVEC-derived EVs enhanced the viability and adhesion of LL29 and HaCaT cells through up-regulation of JMJD3. Next, we showed that JMJD3 overexpression enhanced LL29 and HaCaT cell viability and adhesion through elevating RIPK3, which induced the phosphorylation of AKT during the wound-healing process. We next developed a mouse skin wound model to investigate the actions of miR-106b in vivo after 14 days. The delivery of miR-106b via HUVEC-derived EVs delayed wound healing through suppression of collagen I content and angiogenesis, but had no effects on pro-inflammatory cytokines. In conclusion, miR-106b from HUVEC-derived EVs inhibits JMJD3 and RIPK3, leading to the inhibition of skin wound healing, thus constituting a new therapeutic target.

Qi L ，Lu Y ，Wang Z ，Zhang G ... -  《-》

Extracellular vesicle-carried microRNA-27b derived from mesenchymal stem cells accelerates cutaneous wound healing via E3 ubiquitin ligase ITCH.

Mesenchymal stem cells (MSCs) have been highlighted as promising candidate cells in relation to cutaneous wound healing. The current study aimed to investigate whether MSC-derived extracellular vesicles (EVs) could transfer microRNA-27b (miR-27b) to influence cutaneous wound healing. The miR-27b expression was examined in the established cutaneous wound mouse model, and its correlation with the wound healing rate was evaluated by Pearson's correlation analysis. The identified human umbilical cord MSC-derived EVs were co-cultured with human immortal keratinocyte line HaCaT and human skin fibroblasts (HSFs). The mice with cutaneous wound received injections of MSC-derived EVs. The effects of EVs or miR-27b loaded on wound healing and cellular functions were analysed via gain- and loss-of-function approaches in the co-culture system. Dual-luciferase reporter gene assay was employed to verify the relationship between miR-27b and Itchy E3 ubiquitin protein ligase (ITCH). Rescue experiments were conducted to investigate the underlying mechanisms associated with the ITCH/JUNB/inositol-requiring enzyme 1α (IRE1α) axis. miR-27b was down-regulated in the mouse model, with its expression found to be positively correlated with the wound healing rate. Abundant miR-27b was detected in the MSC-derived EVs, while EV-transferred miR-27b improved cutaneous wound healing in mice and improved proliferation and migration of HaCaT cells and HSFs in vitro. As a target of miR-27b, ITCH was found to repress cell proliferation and migration. ITCH enhanced the JUNB ubiquitination and degradation, ultimately inhibiting JUNB and IRE1α expressions and restraining wound healing. Collectively, MSC-derived EVs transferring miR-27b can promote cutaneous wound healing via ITCH/JUNB/IRE1α signalling, providing insight with clinical implications.

Cheng S ，Xi Z ，Chen G ，Liu K ，Ma R ，Zhou C ... -  《-》

Extracellular vesicle-enclosed miR-486-5p mediates wound healing with adipose-derived stem cells by promoting angiogenesis.

Adipose-derived stem cells (ASC) are said to have a pivotal role in wound healing. Specifically, ASC-secreted extracellular vesicles (EV) carry diverse cargos such as microRNAs (miRNAs) to participate in the ASC-based therapies. Considering its effects, we aimed to investigate the role of ASC-EVs in the cutaneous wound healing accompanied with the study on the specific cargo-medicated effects on wound healing. Two full-thickness excisional skin wounds were created on mouse dorsum, and wound healing was recorded at the indicated time points followed by histological analysis and immunofluorescence staining for CD31 and α-SMA. Human skin fibroblasts (HSFs) and human microvascular endothelial cells (HMECs) were co-cultured with EVs isolated from ASC (ASC-EVs), respectively, followed by the evaluation of their viability and mobility using CCK-8, scratch test and transwell migration assays. Matrigel-based angiogenesis assays were performed to evaluate vessel-like tube formation by HMECs in vitro. ASC-EVs accelerated the healing of full-thickness skin wounds, increased re-epithelialization and reduced scar thickness whilst enhanced collagen synthesis and angiogenesis in murine models. However, miR-486-5p antagomir abrogated the ASC-EVs-induced effects. Intriguingly, miR-486-5p was found to be highly enriched in ASC-EVs, exhibiting an increase in viability and mobility of HSFs and HMECs and enhanced the angiogenic activities of HMECs. Notably, we also demonstrated that ASC-EVs-secreted miR-486-5p achieved the aforesaid effects through its target gene Sp5. Hence, our results suggest that miR-486-5p released by ASC-EVs could be a critical mediator to develop an ASC-based therapeutic strategy for wound healing.

Lu Y ，Wen H ，Huang J ，Liao P ，Liao H ，Tu J ，Zeng Y ... -  《-》

Extracellular vesicles-encapsulated microRNA-29b-3p from bone marrow-derived mesenchymal stem cells promotes fracture healing via modulation of the PTEN/PI3K/AKT axis.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are well-established as vital regulators of fracture healing, whereas angiogenesis is one of the critical processes during the course of bone healing. Accordingly, the current study sought to determine the functions of microRNA (miR)-29b-3p from BM-MSCs-derived extracellular vesicles (EVs) on the angiogenesis of fracture healing via the PTEN/PI3K/AKT axis. Firstly, BM-MSCs-EVs were extracted and identified. The lentiviral protocol was adopted to construct miR-29b-3pKD-BMSCs or miR-negative control-BMSCs, which were then co-cultured with human umbilical vein endothelial cells (HUVECs) in vitro to determine the roles of EVs-encapsulated miR-29b-3p on the proliferation, migration, and angiogenesis of HUVECs in vitro with the help of a CCK-8 assay, scratch test, and tube formation assay. Subsequent database prediction, luciferase activity assay, RT-qPCR, and Western blot assay findings identified the downstream target gene of miR-29b-3p, PTEN, and a signaling pathway, PI3K/AKT. Furthermore, the application of si-PTEN attenuated the effects induced by miR-29b-3pKD-EVs. Finally, a mouse model of femoral fracture was established with a locally instilled injection of equal volumes of BM-MSCs-EVs and miR-29b-3pKD-BM-MSCs-EVs. Notably, the mice treated with BMSC-EVs presented with enhanced neovascularization at the fracture site, in addition to increased bone volume (BV), BV/tissue volume, and mean bone mineral density; whereas miR-29b-3pKD-BMSCs-EVs-treated mice exhibited decreased vessel density with poor fracture healing capacity. Collectively, our findings elicited that BM-MSCs-EVs carrying miR-29b-3p were endocytosed by HUVECs, which consequently suppressed the PTEN expression and activated the PI3K/AKT pathway, thereby promoting HUVEC proliferation, migration, and angiogenesis, and ultimately facilitating fracture healing.

Yang J ，Gao J ，Gao F ，Zhao Y ，Deng B ，Mu X ，Xu L ... -  《-》

Silencing microRNA-210 in Hypoxia-Induced HUVEC-Derived Extracellular Vesicles Inhibits Hemangioma.

Hemangioma (Hem) is a benign tumor commonly seen in infancy with a relative high morbidity. Human umbilical vein endothelial cell (HUVEC)-derived extracellular vesicles (EVs) are actively participated in Hem. Therefore, this study is designed to figure out the underlying mechanism of HUVEC-derived EVs in Hem. Initially, EVs were separated from HUVECs and identified. HUVEC-derived EVs in normoxia or hypoxia were then cultivated with Hem endothelial cells (HemECs) to test the proliferation, apoptosis, and migration of HemECs. Microarray analysis was performed to select microRNAs (miRs) with differential expression. miR-210 in hypoxia-induced HUVECs was silenced, and the relevant EVs were extracted and then co-cultured with HemECs to perform biological effect experiments. Then, the target relation between miR-210 and homeobox A9 (HOXA9) was identified by the dual luciferase reporter gene assay and RNA immunoprecipitation assay. Moreover, xenograft transplantation was also applied to confirm the in vitro experiments. Hypoxia-induced HUVECs promoted release of EVs, which were absorbed by HemECs. Hypoxia-induced HUVEC-EVs promoted HemEC proliferation and migration and inhibited apoptosis. miR-210 from the hypoxia-induced HUVEC-EVs was highly expressed and promoted HemEC growth. Silencing miR-210 expression in the hypoxia-induced HUVEC-EVs suppresses Hem development in vivo. In addition, miR-210 targeted HOXA9. Silencing miR-210 in HUVEC-derived EVs could suppress Hem by targeting HOXA9. This investigation may provide novel insights for Hem treatment.

Ma J ，Tao X ，Huang Y 《-》