Arif Syrine, Larochelle Sébastien, Moulin Véronique J
Centre de recherche en organogénèse expérimentale de l'Université Laval/LOEX, Centre de recherche du CHU de Québec-Université Laval, Quebec, QC, Canada.
Department of Surgery, Faculty of Medicine, Université Laval, Quebec, QC, Canada.
J Cell Commun Signal. 2020 Dec;14(4):427-438. doi: 10.1007/s12079-020-00572-5. Epub 2020 Jul 1.
During the last stages of wound healing, myofibroblasts differentiate mainly from fibroblasts. Myofibroblasts from normal skin wounds (Wmyo) can communicate with its surrounding using secreted factors. They also have the capacity to produce microvesicles (MVs), a type of extracellular vesicles, as mediators of intercellular communication. MVs cargo are potentially capable of regulating the behavior of targeted cells and tissues. The aim of this study is to evaluate the effect of Wmyo-derived MVs on dermal fibroblasts and to determine the responsible signaling molecule. Microvesicles were obtained from culture media of myofibroblasts and characterized using protein quantification, dynamic light scattering and transmission electron microscopy. Uptake of fluorescent MVs in fibroblasts was assessed by flow cytometry. Cytokines concentrations were quantified in MV samples by a multiplex ELISA. Different concentration of MVs or a selected cytokine were used as treatments over fibroblasts culture for 5 days. Following the treatments, parameters linked to the extracellular matrix were studied. Lastly, the selected cytokine was neutralized within MVs before evaluating collagen production. We showed that Wmyo derived-MVs were internalized by dermal fibroblasts. Cytokine array analysis revealed that a large amount of placental growth factor 1 (PLGF-1) (0.88 ± 0.63 pg/μg proteins in MVs) could be detected in MVs samples. Cutaneous fibroblasts treated with MVs or PLGF-1 showed significantly stimulated procollagen I level production (Fold change of 1.80 ± 0.18 and 2.07 ± 0.18, respectively). Finally, the neutralization of PLGF-1 in MVs significantly inhibited the production of procollagen I by fibroblasts. Our study shows that Wmyo derived-MVs are involved in intercellular communication by stimulating collagen production by fibroblasts during wound healing. This effect is possibly attained through PLGF-1 signalling. These findings represent a promising opportunity to gain insight into how MVs and Wmyo may mediate the healing of a skin wound.
在伤口愈合的最后阶段，肌成纤维细胞主要由成纤维细胞分化而来。正常皮肤伤口的肌成纤维细胞（Wmyo）可利用分泌因子与周围环境进行通讯。它们还具有产生微泡（MVs）的能力，微泡是一种细胞外囊泡，作为细胞间通讯的介质。MVs的货物有可能调节靶细胞和组织的行为。本研究的目的是评估Wmyo来源的MVs对真皮成纤维细胞的影响，并确定相关的信号分子。从肌成纤维细胞的培养基中获得微泡，并通过蛋白质定量、动态光散射和透射电子显微镜进行表征。通过流式细胞术评估成纤维细胞中荧光MVs的摄取。通过多重ELISA对MVs样品中的细胞因子浓度进行定量。将不同浓度的MVs或选定的细胞因子用于处理成纤维细胞培养物5天。处理后，研究与细胞外基质相关的参数。最后，在评估胶原蛋白产生之前，将选定的细胞因子在MVs中进行中和。我们发现Wmyo来源的MVs被真皮成纤维细胞内化。细胞因子阵列分析显示，在MVs样品中可检测到大量胎盘生长因子1（PLGF-1）（MVs中为0.88±0.63 pg/μg蛋白质）。用MVs或PLGF-1处理的皮肤成纤维细胞显示原胶原I水平的产生受到显著刺激（分别为1.80±0.18和2.07±0.18倍变化）。最后，MVs中PLGF-1的中和显著抑制了成纤维细胞原胶原I的产生。我们的研究表明，Wmyo来源的MVs通过在伤口愈合过程中刺激成纤维细胞产生胶原蛋白参与细胞间通讯。这种作用可能是通过PLGF-1信号传导实现的。这些发现为深入了解MVs和Wmyo如何介导皮肤伤口愈合提供了一个有前景的机会。
