Wei Pei, Zhong Chenjian, Yang Xiaolan, Shu Futing, Xiao Shichu, Gong Teng, Luo Pengfei, Li Li, Chen Zhaohong, Zheng Yongjun, Xia Zhaofan
Fujian Burn Institute, Fujian Medical University Union Hospital, Fuzhou 350001, Fujian, China.
Department of Burn Surgery, Changhai Hospital, Naval Medical University, Shanghai 200433, China.
Burns Trauma. 2020 Sep 7;8:tkaa020. doi: 10.1093/burnst/tkaa020. eCollection 2020.
Diabetic wounds are one of the most common and serious complications of diabetes mellitus, characterized by the dysfunction of wound-healing-related cells in quantity and quality. Our previous studies revealed that human amniotic epithelial cells (hAECs) could promote diabetic wound healing by paracrine action. Interestingly, numerous studies demonstrated that exosomes derived from stem cells are the critical paracrine vehicles for stem cell therapy. However, whether exosomes derived from hAECs (hAECs-Exos) mediate the effects of hAECs on diabetic wound healing remains unclear. This study aimed to investigate the biological effects of hAECs-Exos on diabetic wound healing and preliminarily elucidate the underlying mechanism.
hAECs-Exos were isolated by ultracentrifugation and identified by transmission electron microscopy, dynamic light scattering and flow cytometry. A series of functional analyses were performed to assess the regulatory effects of hAECs-Exos on human fibroblasts (HFBs) and human umbilical vein endothelial cells (HUVECs) in a high-glycemic microenvironment. High-throughput sequencing and bioinformatics analyses were conducted to speculate the related mechanisms of actions of hAECs-Exos on HFBs and HUVECs. Subsequently, the role of the candidate signaling pathway of hAECs-Exos in regulating the function of HUVECs and HFBs, as well as in diabetic wound healing, was assessed.
hAECs-Exos presented a cup- or sphere-shaped morphology with a mean diameter of 105.89 ± 10.36 nm, were positive for CD63 and TSG101 and could be internalized by HFBs and HUVECs. After that, hAECs-Exos not only significantly promoted the proliferation and migration of HFBs, but also facilitated the angiogenic activity of HUVECs . High-throughput sequencing revealed enriched miRNAs of hAECs-Exos involved in wound healing. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses have shown that the target genes of the top 15 miRNAs were highly enriched in the PI3K-AKT pathway. Further functional studies demonstrated that the PI3K-AKT-mTOR pathway was necessary for the induced biological effects of hAECs-Exos on HFBs and HUVECs, as well as on wound healing, in diabetic mice.
Our findings demonstrated that hAECs-Exos represent a promising, novel strategy for diabetic wound healing by promoting angiogenesis and fibroblast function via activation of the PI3K-AKT-mTOR pathway.
糖尿病伤口是糖尿病最常见且最严重的并发症之一，其特征在于与伤口愈合相关的细胞在数量和质量上存在功能障碍。我们之前的研究表明，人羊膜上皮细胞（hAECs）可通过旁分泌作用促进糖尿病伤口愈合。有趣的是，大量研究表明，源自干细胞的外泌体是干细胞治疗的关键旁分泌载体。然而，源自hAECs的外泌体（hAECs-Exos）是否介导hAECs对糖尿病伤口愈合的影响仍不清楚。本研究旨在探讨hAECs-Exos对糖尿病伤口愈合的生物学作用，并初步阐明其潜在机制。
通过超速离心分离hAECs-Exos，并通过透射电子显微镜、动态光散射和流式细胞术进行鉴定。进行了一系列功能分析，以评估hAECs-Exos在高糖微环境中对人成纤维细胞（HFBs）和人脐静脉内皮细胞（HUVECs）的调节作用。进行高通量测序和生物信息学分析，以推测hAECs-Exos对HFBs和HUVECs的相关作用机制。随后，评估了hAECs-Exos的候选信号通路在调节HUVECs和HFBs功能以及糖尿病伤口愈合中的作用。
hAECs-Exos呈现杯状或球状形态，平均直径为105.89±10.36nm，CD63和TSG101呈阳性，并且可被HFBs和HUVECs内化。此后，hAECs-Exos不仅显著促进了HFBs的增殖和迁移，还促进了HUVECs的血管生成活性。高通量测序揭示了hAECs-Exos中参与伤口愈合的miRNA富集。京都基因与基因组百科全书（KEGG）和基因本体（GO）分析表明，前15个miRNA的靶基因在PI3K-AKT通路中高度富集。进一步的功能研究表明，PI3K-AKT-mTOR通路对于hAECs-Exos诱导的对HFBs和HUVECs以及糖尿病小鼠伤口愈合的生物学作用是必需的。
我们的研究结果表明，hAECs-Exos通过激活PI3K-AKT-mTOR通路促进血管生成和成纤维细胞功能，代表了一种有前景的糖尿病伤口愈合新策略。
