Curing scleroderma (SSc) is now a realistic clinical challenge [3, 4], since its etiology and pathogenesis are unclear. The key clues about its possible mechanisms are limited to vasculopathy, immune dysfunction and fibroblast dysfunction [1, 2]. On the other hand, BMSCs-based therapies, including our previous work, showed some therapeutic effects like attenuating skin fibrosis and apoptosis in BLM-induced SSc model. One of the observations drew our interests that the colonization number and differentiation efficiency of the BMSCs were fewer or lower in the injected sites . It may indicate that the therapeutic mechanism of these BMSCs in treating SSc involves paracrine rather than differentiation. Therefore, in this study, we used BMSCs as a tool to explore the pathogenesis and to develop a potentially more practical treatment for scleroderma, through exploring BMSC-EXOs and their components. Again, BLM-induced SSc in mice was used to mimic the diseases since the pathological changes like increased dermal thickness and collagen accumulation were paralleled with those in the patients with SSc .
The MSCs have been reported to play an anti-fibrotic role in fibrotic diseases such as liver fibrosis , kidney fibrosis , lung fibrosis [31, 32], and skin fibrosis . However, there are still unresolved and unavoidable risks of MSC clinical application such as iatrogenic tumor formation, cellular rejection and infusion toxicity . Also, cells as drugs are still in a very beginning stage and their evaluations and approvals for clinical use has a long way to go. On the other hand, exosomes derived from MSCs have been shown to be the key factors in MSC-to-surrounding cells communication , and are considered as stem cell-based, cell-free drugs and as carrier of siRNA . As compared to BMSC, the BMSC-EXO has several advantages over the cells, like simpler production and storage procedures, and easier to control the quality. The safety of intravenously and intraperitoneally injection of exosome has been verified in animal experiments [36, 37]. Furthermore, the safety and efficiency of MSC-derived exosomes have been evaluated in several clinical trials for various diseases, including inhalation in severe acute respiratory syndrome coronavirus 2 (NCT04276987, ChiCTR2000030261) and intravenous injection in diabetes mellitus (NCT02138331). Therefore, we designed the present study to verify if BMSC-EXO could mediate the effects as BMSCs in treating SSc so that could be developed into a potential therapy without the risks of MSCs. As we expected, subcutaneous BMSC-EXO treatment significantly improved the BLM-induced dermal damage and abnormalities, and reduced the ECM deposition and collagen density. The therapeutic effects of such BMSC-EXOs were as strong as those of the BMSCs. So, BMSC-EXO has great potential to be developed as a new therapy for SSc.
Exosomes serve as carriers that transport functional proteins, mRNAs and miRNAs to various cells, where these factors act as mediators of intercellular communication and signaling pathway [38, 39]. Studies proved that MSC-EXO microRNAs possess the abilities to promote cell proliferation, accelerate injured tissues’ repairment, and inhibit fibrotic diseases [16, 40–42]. In this study, we further demonstrated that there was a series of miRNAs of the BMSC-EXOs, which contributed to the alleviation of SSc through regulating related signaling pathways. Previously, TGFβ pathway, Toll-like receptor signaling and WNT pathway were reported to be the main dysfunctional signaling pathways in the skin of the patients with systemic sclerosis , and TGFβ and Wnt/β-catenin pathways were found hyperactivated to promote ECM production and induce fibrosis [44, 45]. In this study, we focused more on these signal pathways and our data suggested that BMSC-EXO might function through regulating TGFβ and WNT pathways as well as the inflammatory response in treating the BLM-induced SSc. The EXOs inhibited fibroblast activation through down-regulating the expression of TGF-β1 in the dermis, significantly reduced the infiltration of inflammatory cells and inhibit the release of inflammatory factors in the skin of the BLM-induced SSc mice. All these effects were related to the miRNAs of the BMSC-EXOs.
The microRNA let-7 family contains let-7a, 7b, 7c, 7d, 7e, 7f, 7g, 7i and 7j, mir-29, mir-125 and mir-21. They were all found to highly express in the mouse BMSC-EXOs in this study. let-7 is the first discovered microRNA and functionally conserved in vertebrata , and was reported to inhibit production of proinflammatory cytokines such as Il8 and receptors such as Il1r1 and Il23r, to negatively regulate the differentiation of Th17 cell, and to regulate natural killer T cell [47–49]. Both EXOs and microRNAs are closely related to TGFβ signaling pathway when they play regulating roles. For example, the down-regulations of Let-7, mir-29 and mir-30 families in idiopathic pulmonary fibrosis were related to the TGFβ pathway [50, 51], and mir-29 knockdown significantly upregulated the TGFβ signaling in the induction of the pulmonary fibrosis . What’s more, let-7 cooperated with mir-99a and mir125b, both high expressed in BMSC-EXO, when targeted receptor subunits and SMAD signaling transducers to block of the TGFβ pathway . TGFβ signaling and Wnt signaling were found to promote each other to induce the fibrosis in SSc[53, 54]. Mir-21 and mir-29 were reported to target the transducers of Wnt signaling [55, 56]. Thus, addition of microRNAs from BMSC-EXOs could regulate the dysfunctional signals like TGFβ and Wnt to ameliorate the SSc condition include ECM deposition and inflammatory infiltration. This study enriched the understanding on the molecular regulations of exosome mediated microRNAs in the SSc pathogenesis and its treatment with the BMSC-EXOs.