Exosomes released by melanocytes modulate fibroblasts to promote keloid formation: a pilot study

Exosomes released by melanocytes modulate fibroblasts to promote keloid formation: a pilot study 黑色素细胞通过释放外泌体调控成纤维细胞以促进瘢痕疙瘩形成的初步研究 1 equal first-author 2 equal 1 1 corresp Jinghong XU, doctorxjh@zju.edu.cn Jinghong XU, doctorxjh@zju.edu.cn doctorxjh@zju.edu.cn https://orcid.org/0000-0001-5602-4480 Department of Plastic Surgery, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China Department of Plastic Surgery, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China 浙江大学医学院附属第一医院整形外科，中国杭州市，310003 浙江大学医学院附属第一医院整形外科，中国杭州市，310003 Center of Safety Evaluation and Research, Hangzhou Medical College, Hangzhou 310059, China Center of Safety Evaluation and Research, Hangzhou Medical College, Hangzhou 310059, China 杭州医学院安全性评价研究中心，中国杭州市，310059 杭州医学院安全性评价研究中心，中国杭州市，310059 瘢痕疙瘩是常见病理性皮肤愈合，极难预防和根治。临床发现黑色素细胞活性较高的深肤色人群具有瘢痕疙瘩易感性，提示黑色素细胞可能参与其形成过程。瘢痕疙瘩主要由成纤维细胞中TGF-β/Smads通路异常激活所致，而文献报道黑色素细胞分泌物能激活成纤维细胞中TGF-β信号，但机制不明。我们前期研究发现携带特定miRNAs的黑色素细胞外泌体可激活成纤维细胞内TGF-β/Smads通路，从而促进瘢痕疙瘩形成。本研究将从细胞间对话角度揭示深肤色人群易感瘢痕疙瘩的内在机理。 外泌体 黑色素细胞 成纤维细胞 瘢痕疙瘩 miRNAs Keloids are a common type of pathological scar as a result of skin healing, which are extremely difficult to prevent and treat without recurrence. The pathological mechanism of keloids is the excessive proliferation of fibroblasts, which synthesize more extracellular matrices (ECMs), including type I/III collagen (COL-1/3), mucopolysaccharides, connective tissue growth factor (CTGF, also known as cellular communication network factor 2 (CCN2)), and fibronectin (FN) in scar tissue, mostly through the abnormal activation of transforming growth factor-‍β (TGF-‍β)/Smads pathway ( Finnson et al., 2013 Finnson et al., 2013 ; Song et al., 2018 Song et al., 2018 ). Genetic factors, including race and skin tone, are considered to contribute to keloid formation. The reported incidence of keloids in black people is as high as 16%, whereas white people are less affected. The prevalence ratio of colored people to white people is 5:1‍‍–‍‍15:1 ( Rockwell et al., 1989 Rockwell et al., 1989 ; LaRanger et al., 2019 LaRanger et al., 2019 ). In addition, keloids have not been reported in albinism patients of any race, and those with darker skin in the same race are more likely to develop this disease ( LaRanger et al., 2019 LaRanger et al., 2019 ). Skin melanocyte activity is significantly different among people with different skin tones. The more active the melanocyte function, the more melanin is produced and the darker the skin. Similarly, in the same individual, the incidence of keloids increases during periods when melanocytes are active, such as adolescence and pregnancy. Keloids rarely appear in areas where melanocytes synthesize less melanin, such as in the palms and soles. Thus, the formation of keloids seems to be closely related to melanocyte activity. In the skin, melanocytes are located in the basal layer of the epidermis, while fibroblasts are found in the dermis and fascia. The basement membrane is broken when a wound appears, thus enabling fibroblasts that migrate from the fascia to the edge of the wound to communicate with the melanocytes ( Correa-Gallegos et al., 2019 Correa-Gallegos et al., 2019 ). Previous in vitro experiments showed that both the supernatants of cultured melanocytes and co-culturing melanocytes with fibroblasts significantly stimulated proliferation and DNA synthesis in fibroblasts ( Gao et al., 2013 Gao et al., 2013 ). Herein, we observed that melanocytes in keloids are more active in vivo compared to normal scars, since there are more mel anocytes and melanosomes in keloid tissue (Figs. 1 1 a and 1 1 b). During keloid formation, the TGF-‍β type I receptor (TβR-I) directly phosphorylates the downstream substrate Smad2/3, and phosphorylated Smad2/3 (p-Smad2/3) binds to the mediator-type Smad4 to form a trimeric transcription factor that enters the nucleus and participates in promoting the proliferation of fibroblasts and ECM synthesis ( Wang et al., 2012 Wang et al., 2012 ). Moreover, we found that the melanocyte culture supernatant in a suitable concentration can upregulate the TGF-‍β/Smads transduction levels of fibroblasts (Figs. 1 1 c and 1 1 d) and ECM synthesis in vitro (Figs. 1 1 d and 1 1 e). Therefore, functional melanocytes may release some secretions to stimulate fibroblasts during wound healing and promote excessive fibrosis, thereby contributing to the occurrence and development of keloids. Intercellular communication can be carried out through soluble molecules or extracellular vesicles. Exosomes are common types of extracellular vesicles secreted by most cells, which carry the molecular information of parental cells, such as DNA, RNA, and proteins involved in a variety of physiological and pathological processes ( Raposo and Stoorvogel, 2013 Raposo and Stoorvogel, 2013 ; Chen et al., 2020 Chen et al., 2020 ; Geng et al., 2021 Geng et al., 2021 ; Liu et al., 2021 Liu et al., 2021 ). We observed a typical exosomal structure in and around melanocytes in vivo ( Fig. 2 Fig. 2 a). Exosomes from the supernatant of human primary melanocytes were isolated through differential centrifugation and identified by transmission electron microscopy (TEM) observation ( Fig. 2 Fig. 2 a) and western blot analysis ( Fig. 2 Fig. 2 b). The results of nanoparticle tracking analysis confirmed that the sizes of isolated extracellular vesicles (EVs) were in the range of 30‍–‍150 nm. Interestingly, when melanocytes were co-cultured with fibroblasts for a short period, the distribution of exosomes within the melanocytes shifted from around the nucleus to the side near the fibroblasts toward the contact site of cells ( Fig. 2 Fig. 2 c), as quantified by the increased distance of exosomes from the nuclei ( Fig. 2 Fig. 2 c, bottom panel). Similar redistributions have been reported in other exosomal secretion systems ( Mittelbrunn et al., 2011 Mittelbrunn et al., 2011 ; lo Cicero et al., 2015). To verify whether exosomes are the key to melanocytes affecting fibroblasts, eventually leading to keloid formation by activating the TGF-‍β pathway, we carried out further investigation. Exosomes obtained by differential ultracentrifugation of the melanocyte supernatant were resuspended in phosphate-buffered saline (PBS) to obtain an exosome suspension, and the supernatant obtained in the last step of exosome purification was the culture supernatant depleted of melanocyte exosomes. We found that melanocyte-derived exosome suspension could upregulate the TGF-‍β/Smads transduction levels of fibroblasts, promote collagen synthesis, and prevent collagen degradation by inhibiting the secretion of matrix metalloproteinases (MMPs) (Figs. 2 2 d and 2 2 e), whereas the culture supernatants depleted of melanocyte exosomes had no such effect, thereby excluding the role of soluble molecules. Therefore, we speculated that melanocytes could act on adjacent fibroblasts through exosomes and the TGF-‍β signaling pathway to regulate the occurrence and development of keloids. Exosomes can carry microRNAs (miRNAs) to achieve the intracellular transfer of their own RNAs; most miRNAs that play a role in circulation take exosomes as carriers ( Thomou et al., 2017 Thomou et al., 2017 ). For example, macrophage exosomes deliver miR-223 to ovarian cancer cells to induce chemotherapy resistance ( Zhu et al., 2019 Zhu et al., 2019 ), and bone marrow mesenchymal stem cell exosomes transport miR-29b-3p to regulate aging-related insulin resistance ( Su et al., 2019 Su et al., 2019 ). Melanocyte activity is enhanced by ultraviolet B (UVB) irradiation. In our previous study, RNA sequencing showed that 15 miRNAs levels were higher in UVB-irradiated melanocyte-derived exosomes than in the non-irradiated melanocyte-derived exosomes ( Shen et al., 2020 Shen et al., 2020 ). Among them, miR-7704, miR-320a-3p, miR-423-5p, miR-375-3p, and miR-1246 were predicted to target multiple molecules in the TGF-‍β/Smads signaling pathway, including Smad-specific E3 ubiquitin protein ligase 1 (Smurf1), bone morphogenetic protein receptor type 1a (Bmpr1a), Smad9, repulsive guidance molecule A (Rgma), bone morphogenetic protein 8b (Bmp8b), Bmpr1b, gremlin 2 (Grem2), and cysteine-rich transmembrane BMP regulator 1 (Crim1). In addition, miR-423-5p ( Feng et al., 2019 Feng et al., 2019 ), miR-375-3p ( Yang et al., 2019 Yang et al., 2019 ), and miR-1246 ( Liu et al., 2019 Liu et al., 2019 ) were reported to be closely related to fibrosis. Notably, miR-7704 is the top upregulated miRNA among the five selected ones, of which the predicted target Smurf1 is one of the key negative regulators of the TGF‍-‍β/Smads pathway, which has not been reported in the current literature. Smurf proteins (Smurf1/2) can recruit Smad7 to the plasma membrane, thus inhibiting the phosphorylation of Smad2/3 and downregulating the TGF-β pathway. We found that Smurf1, as a key negative regulator, was downregulated by both melanocyte supernatant and exosome suspension (Figs. 1 1 d and 2 2 e). Moreover, melanocyte-derived exosomes obtained after transfection with anti-miR-7704 into melanocytes no longer upregulated the TGF-β/Smads transduction levels and collagen synthesis by fibroblasts ( Fig. 3 Fig. 3 a). The dual-luciferase reporter assay verified the target relationship between miR-7704 and Smurf1 in fibroblasts ( Fig. 3 Fig. 3 b). The results indicated that melanocyte exosomes may secrete miR-7704 to target and inhibit Smurf1 in fibroblasts, thereby achieving the regulation of the TGF-β/Smads pathway. This study demonstrated that melanocyte-derived exosomal miR-7704 promotes keloid formation by inhibiting Smurf1 in fibroblasts and activating the TGF‍-‍β/Smads pathway ( Fig. 4 Fig. 4 ). Our findings not only collectively highlight a novel mode of communication between melanocytes and fibroblasts, attributing the novel function for exosomes to the regulation of keloid formation, but also contribute to revealing the biological nature of keloids prevalent in dark-skinned people from the microenvironmental perspective. Our preliminary study provides data on the function of melanocytes of releasing exosomes during the formation of keloids. All changes in this process, especially the mechanism of exosomes and exosomal miR-7704, need to be further studied. Given this innovative discovery, we look forward to further breakthroughs in this field, as the reassessment of crosstalk between melanocytes and fibroblasts is expected to provide new opportunities for the prevention and treatment of scars and keloids. Materials and methods 1 Detailed methods are provided in the electronic supplementary materials of this paper. References Chen C , Liu JM , Luo YP , 2020 . MicroRNAs in tumor immun ity: functional regulation in tumor-associated macrophages. J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 21 ( 1 ): 12 - 28 . https://doi.org/10.1631/jzus.B1900452 https://doi.org/10.1631/jzus.B1900452 MicroRNAs in tumor immun Correa-Gallegos D , Jiang DS , Christ S , et al. , 2019 . Patch repair of deep wounds by mobilized fascia . Nature , 576 ( 7786 ): 287 - 292 . https://doi.org/10.1038/s41586-019-1794-y https://doi.org/10.1038/s41586-019-1794-y Patch repair of deep wounds by mobilized fascia Feng MH , Li JW , Sun HT , et al. , 2019 . Sulforaphane inhibits the activation of hepatic stellate cell by miRNA-423-5p targeting suppressor of fused . Hum Cell , 32 ( 4 ): 403 - 410 . https://doi.org/10.1007/s13577-019-00264-2 https://doi.org/10.1007/s13577-019-00264-2 Sulforaphane inhibits the activation of hepatic stellate cell by miRNA-423-5p targeting suppressor of fused Finnson KW , McLean S , di Guglielmo GM , et al. , 2013 . Dynamics of transforming growth factor beta signaling in wound healing and scarring . Adv Wound Care (New Rochelle) , 2 ( 5 ): 195 - 214 . https://doi.org/10.1089/wound.2013.0429 https://doi.org/10.1089/wound.2013.0429 Dynamics of transforming growth factor beta signaling in wound healing and scarring Gao FL , Jin R , Zhang L , et al. , 2013 . The contribution of melanocytes to pathological scar formation during wound healing . Int J Clin Exp Med , 6 ( 7 ): 609 - 613 . The contribution of melanocytes to pathological scar formation during wound healing Geng H , Zhou QC , Guo WH , et al. , 2021 . Exosomes in bladder cancer: novel biomarkers and targets . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 22 ( 5 ): 341 - 347 . https://doi.org/10.1631/jzus.B2000711 https://doi.org/10.1631/jzus.B2000711 Exosomes in bladder cancer: novel biomarkers and targets LaRanger R , Karimpour-Fard A , Costa C , et al. , 2019 . Analysis of keloid response to 5-fluorouracil treatment and long-term prevention of keloid recurrence . Plast Reconstr Surg , 143 ( 2 ): 490 - 494 . https://doi.org/10.1097/prs.0000000000005257 https://doi.org/10.1097/prs.0000000000005257 Analysis of keloid response to 5-fluorouracil treatment and long-term prevention of keloid recurrence Liu CM , Liao YW , Hsieh PL , et al. , 2019 . miR-1246 as a therapeutic target in oral submucosa fibrosis pathogenesis . J Formos Med Assoc , 118 ( 7 ): 1093 - 1098 . https://doi.org/10.1016/j.jfma.2019.02.014 https://doi.org/10.1016/j.jfma.2019.02.014 miR-1246 as a therapeutic target in oral submucosa fibrosis pathogenesis Liu CY , Chen SX , Zhang HF , et al. , 2021 . Bioinformatic analysis for potential biological processes and key targets of heart failure-related stroke . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 22 ( 9 ): 718 - 732 . https://doi.org/10.1631/jzus.B2000544 https://doi.org/10.1631/jzus.B2000544 Bioinformatic analysis for potential biological processes and key targets of heart failure-related stroke lo Cicero A , Delevoye C , Gilles-Marsens F , et al. , 2015 . Exosomes released by keratinocytes modulate melanocyte pigmentation . Nat Commun , 6 : 7506 . https://doi.org/10.1038/ncomms8506 https://doi.org/10.1038/ncomms8506 Exosomes released by keratinocytes modulate melanocyte pigmentation Mittelbrunn M , Gutiérrez-Vázquez C , Villarroya-Beltri C , et al. , 2011 . Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells . Nat Commun , 2 : 282 . https://doi.org/10.1038/ncomms1285 https://doi.org/10.1038/ncomms1285 Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells Raposo G , Stoorvogel W , 2013 . Extracellular vesicles: exosomes, microvesicles, and friends . J Cell Biol , 200 ( 4 ): 373 - 383 . https://doi.org/10.1083/jcb.201211138 https://doi.org/10.1083/jcb.201211138 Extracellular vesicles: exosomes, microvesicles, and friends Rockwell WB , Cohen IK , Ehrlich HP , 1989 . Keloids and hypertrophic scars: a comprehensive review . Plast Reconstr Surg , 84 ( 5 ): 827 - 837 . Keloids and hypertrophic scars: a comprehensive review Shen ZR , Sun JQ , Shao JJ , et al. , 2020 . Ultraviolet B irradiation enhances the secretion of exosomes by human primary melanocytes and changes their exosomal miRNA profile . PLoS ONE , 15 ( 8 ): e0237023 . https://doi.org/10.1371/journal.pone.0237023 https://doi.org/10.1371/journal.pone.0237023 Ultraviolet B irradiation enhances the secretion of exosomes by human primary melanocytes and changes their exosomal miRNA profile Song KX , Liu S , Zhang MZ , et al. , 2018 . Hyperbaric oxygen therapy improves the effect of keloid surgery and radiotherapy by reducing the recurrence rate . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 19 ( 11 ): 853 - 862 . https://doi.org/10.1631/jzus.B1800132 https://doi.org/10.1631/jzus.B1800132 Hyperbaric oxygen therapy improves the effect of keloid surgery and radiotherapy by reducing the recurrence rate Su T , Xiao YZ , Xiao Y , et al. , 2019 . Bone marrow mesenchymal stem cells-derived exosomal miR-29b-3p regulates aging-associated insulin resistance . ACS Nano , 13 ( 2 ): 2450 - 2462 . https://doi.org/10.1021/acsnano.8b09375 https://doi.org/10.1021/acsnano.8b09375 Bone marrow mesenchymal stem cells-derived exosomal miR-29b-3p regulates aging-associated insulin resistance Thomou T , Mori MA , Dreyfuss JM , et al. , 2017 . Adipose-derived circulating miRNAs regulate gene expression in other tissues . Nature , 542 ( 7642 ): 450 - 455 . https://doi.org/10.1038/nature21365 https://doi.org/10.1038/nature21365 Adipose-derived circulating miRNAs regulate gene expression in other tissues Wang YW , Ren JH , Xia K , et al. , 2012 . Effect of mitomycin on normal dermal fibroblast and HaCat cell: an in vitro study . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 13 ( 12 ): 997 - 1005 . https://doi.org/10.1631/jzus.B1200055 https://doi.org/10.1631/jzus.B1200055 Effect of mitomycin on normal dermal fibroblast and HaCat cell: an in vitro study Yang YZ , Zhao XJ , Xu HJ , et al. , 2019 . Magnesium isoglycyrrhizinate ameliorates high fructose-induced liver fibrosis in rat by increasing miR-375-3p to suppress JAK2/STAT3 pathway and TGF-β1/Smad signaling . Acta Pharmacol Sin , 40 ( 7 ): 879 - 894 . https://doi.org/10.1038/s41401-018-0194-4 https://doi.org/10.1038/s41401-018-0194-4 Magnesium isoglycyrrhizinate ameliorates high fructose-induced liver fibrosis in rat by increasing miR-375-3p to suppress JAK2/STAT3 pathway and TGF-β1/Smad signaling Zhu XL , Shen HL , Yin XM , et al. , 2019 . Macrophages derived exosomes deliver miR-223 to epithelial ovarian cancer cells to elicit a chemoresistant phenotype . J Exp Clin Cancer Res , 38 : 81 . https://doi.org/10.1186/s13046-019-1095-1 https://doi.org/10.1186/s13046-019-1095-1 Macrophages derived exosomes deliver miR-223 to epithelial ovarian cancer cells to elicit a chemoresistant phenotype Jiaqi SUN performed the experimental research and data analysis. Zeren SHEN designed the experiments and wrote this article. Jinjin SHAO and Jinghong XU gave some good suggestions on the revision of the manuscript, polished English, and checked the final version. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work is supported by the Zhejiang Provincial Natural Science Foundation of China (No. LQ22H150005) and the National Natural Science Foundation of China (No. 81873937). 10.1631/jzus.B2200036 1673-1581（2022）08-0699-06 This work is supported by the Zhejiang Provincial Natural Science Foundation of China (No. LQ22H150005) and the National Natural Science Foundation of China (No. 81873937). 2022-01-17 2022-05-11 2022-08-15 2022-12-27 2022-07-12 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2022 浙江大学出版社 Zeren SHEN, Jinjin SHAO, Jiaqi SUN, and Jinghong XU declare that they have no conflict of interest. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. Additional informed consent was obtained from all patients for whom identifying information is included in this article. Compliance with ethics guidelines Supplementary information 1 Materials and methods 浙江大学学报（英文版）（B辑：生物医学和生物技术） 8 23