Extracellular vesicles from 3D cultured dermal papilla cells improve wound healing via Krüppel-like factor 4/vascular endothelial growth factor A -driven angiogenesis.

IF 6.3 1区 医学 Q1 DERMATOLOGY Burns & Trauma Pub Date : 2023-10-30 eCollection Date: 2023-01-01 DOI:10.1093/burnst/tkad034
Yunwei Wang, Kuo Shen, Yulin Sun, Peng Cao, Jia Zhang, Wanfu Zhang, Yang Liu, Hao Zhang, Yang Chen, Shaohui Li, Chaolei Xu, Chao Han, Yating Qiao, Qingyi Zhang, Bin Wang, Liang Luo, Yunshu Yang, Hao Guan
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Abstract

Background: Non-healing wounds are an intractable problem of major clinical relevance. Evidence has shown that dermal papilla cells (DPCs) may regulate the wound-healing process by secreting extracellular vesicles (EVs). However, low isolation efficiency and restricted cell viability hinder the applications of DPC-EVs in wound healing. In this study, we aimed to develop novel 3D-DPC spheroids (tdDPCs) based on self-feeder 3D culture and to evaluate the roles of tdDPC-EVs in stimulating angiogenesis and skin wound healing.

Methods: To address the current limitations of DPC-EVs, we previously developed a self-feeder 3D culture method to construct tdDPCs. DPCs and tdDPCs were identified using immunofluorescence staining and flow cytometry. Subsequently, we extracted EVs from the cells and compared the effects of DPC-EVs and tdDPC-EVs on human umbilical vein endothelial cells (HUVECs) in vitro using immunofluorescence staining, a scratch-wound assay and a Transwell assay. We simultaneously established a murine model of full-thickness skin injury and evaluated the effects of DPC-EVs and tdDPC-EVs on wound-healing efficiency in vivo using laser Doppler, as well as hematoxylin and eosin, Masson, CD31 and α-SMA staining. To elucidate the underlying mechanism, we conducted RNA sequencing (RNA-seq) of tdDPC-EV- and phosphate-buffered saline-treated HUVECs. To validate the RNA-seq data, we constructed knockdown and overexpression vectors of Krüppel-like factor 4 (KLF4). Western blotting, a scratch-wound assay, a Transwell assay and a tubule-formation test were performed to detect the protein expression, cell migration and lumen-formation ability of KLF4 and vascular endothelial growth factor A (VEGFA) in HUVECs incubated with tdDPC-EVs after KLF4 knockdown or overexpression. Dual-luciferase reporter gene assays were conducted to verify the activation effect of KLF4 on VEGFA.

Results: We successfully cultured tdDPCs and extracted EVs from DPCs and tdDPCs. The tdDPC-EVs significantly promoted the proliferation, lumen formation and migration of HUVECs. Unlike DPC-EVs, tdDPC-EVs exhibited significant advantages in terms of promoting angiogenesis, accelerating wound healing and enhancing wound-healing efficiency both in vitro and in vivo. Bioinformatics analysis and further functional experiments verified that the tdDPC-EV-regulated KLF4/VEGFA axis is pivotal in accelerating wound healing.

Conclusions: 3D cultivation can be utilized as an innovative optimization strategy to effectively develop DPC-derived EVs for the treatment of skin wounds. tdDPC-EVs significantly enhance wound healing via KLF4/VEGFA-driven angiogenesis.

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来自3D培养的真皮乳头细胞的细胞外小泡通过Krüppel样因子4/血管内皮生长因子A驱动的血管生成改善伤口愈合。
背景:不愈合的伤口是一个棘手的问题,具有重要的临床意义。有证据表明,毛乳头细胞(DPCs)可能通过分泌细胞外小泡(EVs)来调节伤口愈合过程。然而,低的分离效率和有限的细胞活力阻碍了DPC-EVs在伤口愈合中的应用。在本研究中,我们旨在开发基于自饲养3D培养的新型3D-DPC球体(tdDPCs),并评估tdDPC-EVs在刺激血管生成和皮肤伤口愈合中的作用。方法:为了解决目前DPC-EVs的局限性,我们之前开发了一种自饲养三维培养方法来构建TDDPC。用免疫荧光染色和流式细胞术鉴定DPCs和tdDPCs。随后,我们从细胞中提取EVs,并使用免疫荧光染色、划痕试验和Transwell试验在体外比较DPC-EVs和tdDPC-EVs对人脐静脉内皮细胞(HUVECs)的影响。我们同时建立了全层皮肤损伤的小鼠模型,并使用激光多普勒以及苏木精和伊红、Masson、CD31和α-SMA染色评估了DPC-EVs和tdDPC-EVs对体内伤口愈合效率的影响。为了阐明潜在的机制,我们对tdDPC EV和磷酸盐缓冲盐水处理的HUVECs进行了RNA测序(RNA-seq)。为了验证RNA-seq数据,我们构建了Krüppel样因子4(KLF4)的敲除和过表达载体。进行蛋白质印迹、划痕试验、Transwell试验和小管形成试验,以检测KLF4和血管内皮生长因子a(VEGFA)在敲低或过表达后与tdDPC-EVs孵育的HUVECs中的蛋白质表达、细胞迁移和管腔形成能力。进行双荧光素酶报告基因测定以验证KLF4对VEGFA的激活作用。结果:我们成功地培养了tdDPCs,并从DPCs和tdDPCs中提取了EVs。tdDPC-EVs显著促进HUVECs的增殖、管腔形成和迁移。与DPC-EVs不同,tdDPC-EVs在体外和体内都表现出促进血管生成、加速伤口愈合和提高伤口愈合效率的显著优势。生物信息学分析和进一步的功能实验证实,tdDPC-EV调节的KLF4/VEGFA轴在加速伤口愈合方面至关重要。结论:3D培养可作为一种创新的优化策略,有效开发用于治疗皮肤伤口的DPC衍生EVs。tdDPC-EVs通过KLF4/VEGFA驱动的血管生成显著增强伤口愈合。
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来源期刊
Burns & Trauma
Burns & Trauma 医学-皮肤病学
CiteScore
8.40
自引率
9.40%
发文量
186
审稿时长
6 weeks
期刊介绍: The first open access journal in the field of burns and trauma injury in the Asia-Pacific region, Burns & Trauma publishes the latest developments in basic, clinical and translational research in the field. With a special focus on prevention, clinical treatment and basic research, the journal welcomes submissions in various aspects of biomaterials, tissue engineering, stem cells, critical care, immunobiology, skin transplantation, and the prevention and regeneration of burns and trauma injuries. With an expert Editorial Board and a team of dedicated scientific editors, the journal enjoys a large readership and is supported by Southwest Hospital, which covers authors'' article processing charges.
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