Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Alleviate Peritoneal Dialysis-Associated Peritoneal Injury

Abstract

Peritoneal fibrosis is a critical sequela that limits the application of peritoneal dialysis (PD). This study explored the role and mechanism of bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) in preventing PD-associated peritoneal injury. C57BL/6 mice were randomized into three groups: a control (saline), peritoneal injury [2.5% glucose peritoneal dialysate + lipopolysaccharide (LPS)], and peritoneal injury + exosome group. After 6 weeks, mice were dissected, and the parietal peritoneum was collected. The level of peritoneal structural and functional damage was assessed. Additionally, transcriptome analysis of the peritoneum and miRNA sequencing on BMSC-Exos were performed. The parietal peritoneum had significantly thickened, and peritoneal function was impaired in the peritoneal injury group. Peritoneal structural and functional damage was significantly reduced after exosome treatment, while peritoneal inflammation, fibrosis, angiogenesis, and mesothelial damage significantly increased. Transcriptomic analysis showed that the BMSC-Exos affected the cell cycle process, cell differentiation, and inflammatory response regulation. Significant pathways in the exosome group were enriched by inflammation, immune response, and cell differentiation, which constitute a molecular network that regulates the peritoneal protective mechanism. Additionally, inflammatory factors (TNF-α, IL-1β), fibrosis markers (α-SMA, collagen-III, fibronectin), profibrotic cytokines (TGF-β1), and angiogenesis-related factor (VEGF) were downregulated at the mRNA and protein levels through BMSC-Exos treatment. BMSC-Exos treatment can prevent peritoneal injury by inhibiting peritoneal fibrosis, inflammation, and angiogenesis, showing a multitarget regulatory effect. Therefore, BMSC-Exos therapy might be a new therapeutic strategy for treating peritoneal injury.

Introduction

Currently, approximately two million people worldwide have end-stage renal disease, and the increasing number of cases each year represents an important health concern [1]. Peritoneal dialysis (PD), an essential therapy for kidney failure, accounts for ∼11% of all modalities of renal replacement treatment worldwide [2]. Recent studies have proven that PD is safe and as efficient as hemodialysis [3,4]. However, peritoneal fibrosis, a serious sequela that limits the application of PD techniques, results from persistent exposure of PD fluids and recurrent episodes of peritonitis. PD induces lasting exposure of the peritoneum to bioincompatible hypertonic dialysis solutions, leading to chronic peritoneal inflammation. Moreover, PD patients are at high risk for infectious peritonitis, which promotes chronic inflammation, peritoneal mesothelial injury, neoangiogenesis, and peritoneal fibrosis. Long-term PD causes the development of structural and functional alterations in the peritoneum, especially peritoneal fibrosis, which is the major contributor to technical failure in PD patients [5 –7].

Methods for clinical prevention of PD-associated peritoneal fibrosis include more biocompatible PD solutions, peritoneal short resting, tamoxifen, heparin, and RAS blockers [5], but their clinical effects are unsatisfactory. Therefore, effective treatment measures for peritoneal fibrosis are lacking. Novel strategies to effectively inhibit peritoneal injury and peritoneal fibrosis are necessary to improve the prognosis of dialysis patients.

Recently, new biological therapies have garnered attention. Mesenchymal stem cell (MSC) therapy alleviates peritoneal injury in animal models by inhibiting TGF-β1 signaling and suppressing inflammation, and may be a potential strategy to prevent peritoneal fibrosis [8,9]. Meanwhile, more and more studies have observed that MSC possess therapeutic capabilities primarily based on their paracrine mechanism [10,11]. The paracrine effects act through the transfer of extracellular vesicles (EVs) (including exosomes and microvesicles) released by MSCs [12]. MSC-derived exosomes (MSC-Exos) are typically 30–150 nm in diameter and cup shaped or round [13]. They are responsible for the horizontal transfer of mRNA, microRNA, and proteins and are integral components of intercellular communication [14]. Exosomes are ideal therapeutic agents owing to their small size, low immunogenicity, long-circulating half-life, good penetration, and good biocompatibility [15].

MSC-Exos could inhibit chronic tissue inflammation and suppress organ fibrosis. Bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) were potent at reducing the chronic damage and fibrosis of kidneys in a rat model of chronic kidney disease [16]. BMSC-Exos attenuate rat myocardial fibrosis both in vivo and in vitro through autophagy activation [17]. Human MSC-derived exosomes inhibited liver fibrosis in a mouse model [18,19]. Delivery of BMSC-Exos could relieve atrial fibrillation-induced rat myocardial fibrosis, apoptosis, and inflammation [20]. To the best of our knowledge, no study has focused on the use of MSC-derived exosomes to reduce peritoneal fibrosis progression during PD. We aimed to elucidate the therapeutic effect of MSC-Exos on peritoneal injury.

Materials and Methods

Induction of peritoneal injury and exosome treatment

Eight-week-old male C57BL/6 mice (Siberfo Biotechnology, Beijing, China) were randomly divided into three groups (n = 6 per group): control, peritoneal injury, and exosome. In the control group, mice were subjected to intraperitoneal (i.p.) saline injection with 0.1 mL/g body weight, daily for 6 weeks. In the peritoneal injury group, mice received i.p. injection with 2.5% PDF (0.1 mL/g, daily, Huaren Pharmaceutical, H20113091) and LPS (10 mg/kg, once/5 days; Sigma, L4130) for 6 weeks [21]. In the exosome group, mice received i.p. injection with 2.5% PD fluid and LPS for 6 weeks, and treatment with BMSC-Exos (200 μg/kg/dose) at the end of the fourth (day 28) and fifth (day 35) weeks. After 6 weeks, all mice were sacrificed through cervical dislocation. Subsequently, samples of parietal peritoneum, serum, and PD effluent were collected.

In the experiments described above, the dosage (200 μg/kg) and timing (day 28 and 35) of BMSC-EXos treatment were selected based on the results of previous experiments (Supplementary Fig. S1). Animal experiments were approved and conducted in accordance with the guidelines of the Animal Ethics Committee of Army Medical University (AMUWEC20191679).5

Isolation and identification of BMSC-Exos

BMSCs from normal mice were isolated and cultured using the whole bone marrow culture method. BMSCs were identified through flow cytometry analysis of BMSC markers and the method of induced differentiation into adipocytes and osteocytes. According to the manufacturer's instructions, exosomes from the BMSC supernatant were isolated and purified utilizing the Exosome Concentration Kit (Rengen Biosciences; EXOCCon10–10) and the Exosome Purification Kit (Rengen Biosciences; EXOSEC0.5–5). BMSC-Exos were identified through western blotting analysis of exosome biomarker proteins and transmission electron microscopy Fig. 1A–D.

FIG. 1.

Identification and in vivo tracking of BMSC-derived exosomes. (A) Morphological observation using light microscopy of BMSC, adipogenic differentiation, and osteogenic differentiation. Scale bar: 100 μm. (B) Flow cytometry analysis of BMSC markers (CD29⁺, CD44⁺, CD34⁻, and CD11b⁻). (C) Representative electron micrograph of cup-shaped exosomes. Scale bar, 200 nm. (D) Western blotting analysis of exosome biomarker proteins (CD9, CD63, and TSG101). (E) Flow diagram of the experiment. (F) Representative histological images from PAS staining of parietal peritoneum of mice at 42 days. Scale bar: 200 μm. (G) The exosome-tracing experiment was implemented with BMSC-derived exosomes, pretreated using DiI cell membrane dye. DiI-labeled exosomes (DiI-Exos) were injected intraperitoneally in the peritoneal injury group for 48 h. Collagen-I represents fibroblasts and myofibroblasts; vimentin represents fibroblasts and peritoneal mesothelial cells. Scale bar: 50 μm. PAS, periodic acid Schiff; BMSC-Exos, bone marrow mesenchymal stem cell-derived exosomes.

Transcriptome sequencing and analysis

RNA-seq analysis was conducted on transcriptome mRNA sequencing (RNA-seq) RNA isolated from mouse parietal peritoneum. According to the manufacturer's protocol, RNA isolation and purification of total samples were performed utilizing TRIzol reagent (Invitrogen). Paired-end (2 × 150 bp) sequencing reads of libraries were conducted on Illumina NovaSeq™ 6000 sequencing platforms. Differentially expressed genes (DEGs) defined as P < 0.05 and fold change (FC) >2 or <0.5 were screened using the DESeq2 (version 1.36.0) [22] and analyzed through Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.

miRNA sequencing

RNA samples from the BMSC-Exos were isolated according to the manufacturer's protocol. miRNA profiling was determined using the QIAseq miRNA Library Kit. Three databases: miRbase, piRNAbank, and Rfam, were compared and quantified. The normalization for each gene unique molecular index count was determined by counts per million, which was calculated using the edgeR package [23].

In vivo tracking of BMSC-Exos

For in vivo tracking, BMSC-Exos were stained with DiI dye (Invitrogen; C7001) according to the manufacturer's protocol. PBS or DiI dye-labeled exosomes (DiI-Exos) were administered through i.p. injection in mice with peritoneal injury. After 48 h, the mice were dissected, and the parietal peritoneal tissue was taken for immunofluorescence (IF) staining. The distribution of fluorescence images of DiI-Exos in the mice's peritoneum were acquired at 553 and 570 nm excitation and emission wavelengths under confocal microscopy (SP8; Leica).

Histology, immunohistochemistry, and IF

Paraffin sections of mouse peritoneum were processed for Periodic Acid Schiff (PAS) and Masson staining. Alpha-smooth muscle actin (α-SMA), fibronectin (FN), collagen-III, myeloperoxidase (MPO), F4/80, CD68, CA125, Griffonia simplicifolia lectin 1 (GS1-lectin), and vascular endothelial growth factor (VEGF) were detected through immunohistochemical staining of paraffin sections. E-Cadherin and Ki-67 were detected through IF staining of frozen sections. The positive staining areas (α-SMA, FN, collagen-III, GS1-lectin, and VEGF) and the percentage of positive cells (MPO, F4/80, and CD68) in the submesothelial area, were determined and measured using lumina v.2.20 (Mitani) and ImageJ v.1.53e (NIH) at eight random fields ( × 200). Antibody information is provided in Supplementary Table S1.

Real-time qPCR

The expression of mRNA was assessed by real-time quantitative PCR (RT-qPCR). RNA isolation from parietal peritoneum samples was performed using TRIzol reagent. Reverse transcription of RNA to cDNA was performed using the RT-PCR Kit (TaKaRa). We used the GoTaq^® qPCR Kit (Promega) to perform RT-qPCR and CFX ManagerTM v.3.1 (Bio-Rad) to analyze the mRNA levels using the 2^−ΔΔCT method. The reference gene GAPDH was used to normalize the PCR results. The sequences of primers are presented in Supplementary Table S2.

Peritoneal equilibration test

The peritoneal permeability and ultrafiltration of mice were measured through a 2-h modified peritoneal equilibration test (PET) on the last day before they were dissected [24]. Two hours after i.p. injection with 3 mL of 7% glucose PD fluid, peritoneal effluents and serum were harvested. The net ultrafiltration volume was calculated as the volume of the peritoneal effluent after 2 h minus the injection volume. We calculated the D/D0 glucose ratio (ratio of dialysate glucose at 2 and 0 h) and the D/P blood urea nitrogen (BUN) ratio (ratio of dialysate BUN to plasma BUN at 2 h) to assess the functional status of peritoneal solute transport.

Statistical analysis

Data are presented as mean ± standard deviations (SD). One-way ANOVA was used for multiple comparisons of parametric variables. The Kruskal–Wallis test was used for multiple comparisons of nonparametric variables. Statistical analysis was conducted by SPSS v.22.0 (IBM).

Results

BMSC-Exos alleviate mouse peritoneal fibrosis induced by 2.5%PDF+LPS

Chronic peritoneal injury is characterized by peritoneal fibrosis, inflammation, and neoangiogenesis [25]. In the process of constructing peritoneal injury mouse model, we found that the peritoneum began to thicken on day 28 with obvious differences on day 42 in the peritoneal injury group (Supplementary Fig. S1A and Fig. 2A, B). Then, we identified the exosomes isolated from BMSCs based on the classic markers (CD9, CD63, and TSG101) and TEM (Fig. 1A–D). To determine the optimal therapeutic dose of BMSC-Exos, we preliminarily proposed three doses of 100, 200, and 400 μg/kg/dose [26,27], and chose day 28 when the peritoneal thickening begins to appear as the treatment time point. The results showed that both 200 and 400 μg/kg/dose could alleviate obvious peritoneal thickening, and no significant differences were noted between the two groups (Supplementary Fig. S1B, C). Next, we aimed at optimizing the timing of BMSC-Exos treatment.

FIG. 2.

BMSC-derived exosomes alleviate peritoneal fibrosis and protect peritoneal function. (A) Representative histological images from Masson's Trichrome staining and immunohistochemical staining of parietal peritoneum for α-SMA, FN, and collagen-III in vivo. Scale bar: 100 μm. (B) Quantitative analysis of the peritoneal submesothelial thickness of mice. Data are the mean ± SD (n = 6). (C–E) Quantitative analysis of the results in Fig. 1A. Data are the mean ± SD (n = 3–5). (F–I) RT-qPCR analysis of α-SMA, TGF-β1, FN, and collagen-III. (J–L) Peritoneal ultrafiltration (J) and peritoneal permeability (K, L) tested by modified peritoneal equilibration test. Data are the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

The results suggested that BMSC-Exos can reduce peritoneal thickening at day 28 and 35 (Supplementary Fig. S1D–F). It was further found that 28 days +35 days' treatment group had better effect on alleviating peritoneal thickening than the other two single treatment groups (Supplementary Fig. S1G, H). Therefore, we chose to treat the mice with 200 μg/kg/dose BMSC-Exos at day 28 and 35. We verified that, i.p. injection of BMSC-Exos could significantly inhibit peritoneal thickening in mice (Figs. 1E, F and 2A, B).

Fibrosis is distinguished by the exceeding deposition and remodeling of the extracellular matrix, of which markers include FN and collagen-III [28]. Furthermore, α-SMA is widely used as a marker for myofibroblasts, which drive fibrosis [29]. The protein and mRNA levels of fibrotic markers (FN, collagen-III, and α-SMA) in the exosome group were markedly reduced compared with those in the peritoneal injury group (Fig. 2A, C–H). Moreover, the mRNA level of profibrotic cytokine TGF-β1 decreased significantly after BMSC-Exos treatment (Fig. 2I). These results indicated that BMSC-Exos treatment alleviated PD-related peritoneal fibrosis in mice.

BMSC-Exos improve functional decline of peritoneal membrane

Peritoneal fibrosis can induce membrane functional failure, resulting in patients' discontinuation of PD treatment [30]. The ultrafiltration of the peritoneal membrane in mice was measured using modified PET. The net ultrafiltration volume in the peritoneal injury group was significantly lower compared with that in the control group, suggesting its ultrafiltration function was impaired (Fig. 2J). Additionally, the peritoneum in mice with peritoneal injury showed a high solute transport; that is, the ratio of glucose concentration (D/D0) decreased, and the ratio of urea nitrogen concentration (D/P BUN) increased significantly (Fig. 2K, L). Exosome treatment mitigated the reduced peritoneal ultrafiltration resulting from peritoneal injury and alleviated the rapid solute transport in the peritoneum (Fig. 2J–L).

BMSC-Exos relieve peritoneal inflammation and angiogenesis

Long-term PD causes peritoneal inflammation and neoangiogenesis, which are crucial pathologic mechanisms during the progression of peritoneal fibrosis [31]. Furthermore, macrophages and neutrophils are the main immune cells that invade the peritoneum during peritonitis [32]. We found that BMSC-Exos treatment suppressed macrophage (CD68⁺ or F4/80⁺) and neutrophil (MPO⁺) infiltration in the peritoneum (Fig. 3A–D). Similarly, the mRNA expression of inflammatory factors (TNF-α, IL-1β, and IL-6) in the exosome group decreased significantly from that of the peritoneal injury group (Fig. 3E–G). Likewise, the expression level of angiogenesis markers (GS1-lectin and VEGF) in the peritoneal tissues of mice in the exosome group was significantly lower compared with those in the peritoneal injury group (Fig. 3H–K). These results suggested that BMSC-Exos attenuated peritoneal inflammation and angiogenesis during peritoneal injury.

FIG. 3.

BMSC-Exos relieve peritoneal inflammation, angiogenesis, and the injury of peritoneal mesothelial cells. (A) Representative histological images from immunohistochemical staining for CD68, F4/80, and MPO in vivo. F4/80 and CD68 represent macrophages; MPO represents neutrophils. Scale bar: 100 μm. (B–D) Quantitation of the results in (A). Data are the mean ± SD (n = 3). (E–G) RT-qPCR analysis of IL-1β, IL-6, and TNF-α mRNAs in mouse parietal peritoneum. Data are the mean ± SD (n = 3). (H) Representative histological images from immunohistochemical staining for GS1-lectin and VEGF in vivo. GS1-lectin and VEGF represent angiogenesis. Scale bar: 100 μm. (I, J) Quantitation of the results in (H). Data are the mean ± SD (n = 3). (K) RT-qPCR analysis of VEGF mRNA in mouse parietal peritoneum. Data are the mean ± SD (n = 3). (L) Representative histological images from immunohistochemical staining for CA125 (scale bar: 100 μm), and immunofluorescence for E-Cadherin (green) and Ki-67 (red) staining in vivo (scale bar: 50 μm). CA125 represents peritoneal mesothelial cells. Black arrowheads indicate the shedding and loss of peritoneal mesothelial cells. E-Cadherin represents peritoneal mesothelial cells; Ki-67 represents cell proliferation. White arrowheads indicate the proliferation of peritoneal mesothelial cells. (M) RT-qPCR analysis of CA125 mRNA in mouse parietal peritoneum. Data are the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

BMSC-Exos alleviate peritoneal mesothelial cell injury

Peritoneal mesothelial cells (PMC) are epithelial-like cells lining the entire abdominal cavity and are the first line of defense, playing a dominant role in peritoneal function [33]. Compared with that in the peritoneal injury group, the expression of CA125, a PMC marker in the mesothelial cell monolayer, significantly increased in the exosome group (Fig. 3L, M). The IF costaining of PMC marker (E-cadherin) and cell proliferation marker (Ki-67) showed that the number of proliferative PMC in the exosome group increased significantly compared with that in the peritoneal injury group (Fig. 3L). Therefore, BMSC-Exos treatment might promote the proliferation and repair of PMC, and consequently, alleviate peritoneal mesothelial layer damage.

BMSC-Exos alleviate peritoneal injury by regulating multiple biological effects

We performed in vivo tracking of BMSC-Exos using DiI dye to determine whether BMSC-Exos can actively penetrate the peritoneal mesothelial layer and submesothelial layer to exert protective effects. The fluorescence signal of DiI-Exos was found in both the peritoneal mesothelial layer and the submesothelial layer, especially in PMC (vimentin+) and submesothelial fibroblasts (collagen-I+) (Fig. 1G).

Transcriptomics was used to detect the influence of exosomes on the whole-genome expression of the peritoneum to clarify the biological role of exosomes in protecting against peritoneal injury. We performed comparisons to identify the DEGs according to the screening criteria (P < 0.05, FC ≥2 or ≤0.5). Of the 55,401 genes detected, 1,686 upregulated and 5,465 downregulated DEGs were found in the exosome group compared with the peritoneal injury group (Fig. 4C). The heat map showed that among the DEGs coregulated by the peritoneal injury effect and BMSC-Exos effect, most were upregulated in the peritoneal injury group (72.8%), whereas the BMSC-Exos treatment significantly inhibited the expression enhancement of these genes (Fig. 4F). We speculated that BMSC-Exos might restore the mRNA expression of most stimulated genes to baseline levels. Furthermore, the marker genes related to inflammation, fibrosis, cell differentiation, and angiogenesis were modulated after BMSC-Exos treatment, consistent with prior results (Fig. 4G–J).

FIG. 4.

BMSC-derived exosomes alleviate peritoneal injury by regulating multiple biological effects. (A) The correlation analysis was performed for transcriptomes of mouse parietal peritoneum among the groups of mice. (B) Venn diagram of overlapping and unique differential genes among the groups of mice. (C) Volcano plots of differentially expressed genes among the groups of mice. Blue dots indicate downregulated differential genes, and red dots indicate upregulated differential genes (P value <0.05). (D, E) Biological process enrichment map of functionally grouped, significantly enriched GO terms of the significantly altered genes, following comparisons of peritoneal injury versus control (D) or comparisons of exosome versus peritoneal injury (E). Representative labels show the major clusters of biological processes in each group. Node size indicates significance of functional enrichment (All P < 0.05). (F) Heat-map of top 50 genes differentially expressed among the groups of mice from peritoneal transcriptomics. Red: increased expression; blue: decreased expression. (G–J) Differential FPKM expression of critical genes related to fibrosis, inflammation, cell differentiation, and angiogenesis among the groups of mice. *P < 0.05; **P < 0.01; ***P < 0.001. GO, gene ontology.

GO enrichment analysis showed that the peritoneal injury effect involves numerous biological processes, mainly cell cycle, cell communication, response to stimulus, and cell differentiation (Fig. 4D). Notably, the BMSC-Exos effect could regulate all the above processes. Additionally, exosome treatment enriched different biological process clusters, such as inflammatory response, regulation of transmembrane transport, and hormone metabolic process (Fig. 4E).

KEGG pathway analysis revealed 152 significantly differential pathways between the exosome and peritoneal injury groups. These pathways were mainly enriched in inflammation (eg, NF-κB pathway, tumor necrosis factor pathway, and chemokine signaling pathway), cell differentiation (eg, Rap1, RAS, and PI3K-Akt signaling pathway), and immune response (eg, antigen processing and presentation, and Th17 cell differentiation) (Supplementary Table S3). Moreover, the exosome group's enrichment score on inflammation, immunity, and cell differentiation, among other related molecular pathways, was higher than that in the peritoneal injury group (Supplementary Fig. S2C, D). In this context, we speculated that BMSC-Exos regulated various biological functions of the peritoneum through multiple pathways.

BMSC-Exos protect the peritoneum through miRNA clusters of different functions

Numerous studies have confirmed that exosomes exert various tissue-protective effects through miRNAs [34 –37]. Through miRNA sequencing, we identified 1960 miRNAs contained in BMSC-Exos. Furthermore, these miRNAs have proved to regulate biological processes, including inflammation, fibrosis, angiogenesis, cell proliferation and differentiation, as well as oxidative stress (Table 1). To reflect the functions of these miRNAs further, a miRNA functional network was constructed based on GO analysis (Fig. 5C). The miRNA-GO network identified that miRNA clusters in BMSC-Exos were involved in regulating functions, such as inflammatory response, cell differentiation, cell proliferation, oxidative stress, and angiogenesis (Fig. 5C). Furthermore, KEGG analysis showed the most differentially expressed miRNAs associated with signaling pathways were related to cell survival (Ras, mTOR, and apoptosis), cell differentiation (Wnt and MAPK), and inflammatory response (TNF) (Fig. 5D). Therefore, BMSC-Exos could present various protective effects during peritoneal injury through a variety of miRNAs with different functions.

FIG. 5.

Biological processes and pathways targeted by miRNAs in BMSC-derived exosomes. (A) Correlation of biological replicates of miRNA read counts of BMSC-derived exosomes. (B) Top 50 miRNAs of total miRNAs present in BMSC-derived exosomes. (C) GO enrichment map of miRNAs in BMSC-derived exosomes targeted multiple biological processes. Nodes represent significantly enriched pathways and the miRNAs that target them. (D) Top 20 KEGG enriched pathways ranked from highest to lowest based on significance.

Table 1.

miRNAs Derived from Bone Marrow Mesenchymal Stem Cell-Derived Exosomes Involved in Important Biological Functions

Biological functions	miRNAs derived from BMSC-Exos
Anti-inflammatory effects	mmu-miR-21a-5p [48], mmu-miR-27b-3p [49], mmu-miR-132-3p [50], mmu-miR-146a-5p [51], mmu-miR-223-3p [52], mmu-miR-129-5p [53], mmu-miR-22-3p [54], mmu-miR-223-3p [55], mmu-miR-542-3p [56], mmu-miR-199a-3p [57], mmu-miR-25-3p [58], mmu-miR-9-5p [59]
Antifibrosis effects	mmu-miR-34c-5p [60], mmu-miR-466f-3p [61], mmu-miR-196b-5p [62], mmu-miR-129-5p [63], mmu-miR-29b-3p [64], mmu-miR-125b-5p [65], mmu-miR-146a-5p [66], mmu-miR-877-3p [67], mmu-miR-27a-3p [68], mmu-miR-130a-3p [69], mmu-miR-212-5p [70], mmu-miR-150-5p [71], mmu-miR-182-5p [72]
Angiogenesis effects	mmu-miR-130a-3p [34], mmu-miR-133a-3p [73], mmu-miR-221-3p [74], mmu-miR-135b-5p [75], mmu-miR-150-5p [76], mmu-miR-153-3p [77], mmu-miR-17-5p [78], mmu-miR-340-5p [79], mmu-miR-195a-3p [80]
Cell proliferation and differentiation effects	mmu-miR-183-5p [81], mmu-miR-6924-5p [82], mmu-miR-877-3p [67], mmu-miR-135a-5p [83], mmu-miR-338-5p [84], mmu-miR-542-3p [85], mmu-miR-369-5p [86], mmu-miR-199a-3p [87], mmu-miR-30a-5p [87], mmu-miR-31-5p [87], mmu-miR-377-3p [87], mmu-miR-330-5p [87], mmu-miR-2861 [87], mmu-miR-200a-3p [87],mmu-miR-125a-3p [87], mmu-miR-384-5p [87], mmu-miR-149-5p [87], mmu-miR-132-3p [87], mmu-miR-590-5p [87], mmu-miR-143-3p [87], mmu-miR-193b-3p [87], mmu-miR-335-5p [87], mmu-miR-140-5p [87], mmu-miR-574-3p [87], mmu-miR-92a-3p [88], mmu-miR-125b-5p [65], mmu-miR-335-5p [89], mmu-miR-129-5p [90], mmu-miR-675-5p [91], mmu-miR-223-3p [92], mmu-miR-199b-5p [93], mmu-miR-140-5p [94], mmu-miR-409-3p [95], mmu-miR-574-3p [96], mmu-let-7b-5p [97], mmu-miR-26b-3p [98], mmu-miR-221-5p [99], mmu-miR-362-3p [100], mmu-miR-425-5p [101], mmu-miR-155-5p [102], mmu-miR-34b-3p [103], mmu-miR-301b-3p [104]
Oxidative stress effects	mmu-miR-140-3p [105], mmu-miR-193a-5p [106], mmu-miR-22-3p [54], mmu-miR-9-5p [59], mmu-miR-129-5p [63], mmu-miR-17-3p [107], mmu-miR-98-5p [108], mmu-miR-133b-3p [109], mmu-miR-100-5p [110], mmu-miR-181a-5p [111], mmu-miR-329-3p [112], mmu-miR-125a-5p [113], mmu-miR-183-5p [114], mmu-miR-338-5p [115]

BMSC-Exos, bone marrow mesenchymal stem cell-derived exosomes.

Discussion

In this study, novel results were obtained showing that PD-associated peritoneal injury in mice can be treated by i.p. injection of BMSC-Exos. BMSC-Exos also alleviated other peritoneal pathological changes, including peritoneal inflammation, PMC injury, and angiogenesis. Transcriptome results showed that the biological processes regulated by BMSC-Exos included inflammatory response, cell differentiation, cell cycle, and response to stimulus. The exosomes contained many miRNAs, which might regulate various genes that could prevent inflammation and fibrosis. Therefore, BMSC-derived exosomes might have various biological therapeutic effects on PD-associated peritoneal injury. Furthermore, i.p. injection of BMSC-Exos could be a new therapeutic strategy for treating peritoneal fibrosis.

In patients undergoing maintenance PD, repeated episodes of peritonitis represent not only a serious complication but also poor prognosis and mortality [38]. In this study, peritonitis was induced using LPS i.p. injections, which accelerated peritoneal fibrosis formation. Peritoneal inflammation and peritoneal fibrosis were observed in mice after 6 weeks. Macrophages are a primary group of resident immune cells in the peritoneum and, simultaneously, the predominant immune population found in PD effluent [39]. In vivo, MSCs relieve PD-induced peritoneal inflammation by regulating macrophage polarization [40]. In vitro, MSC-derived EVs also efficiently exerted anti-inflammatory effects through modulating macrophage phenotype switching [41]. The EVs secreted by MSCs are essentially the exosomes secreted by MSCs. Promoting the phenotypic transformation of macrophages is only one function of exosomes. We found that BMSC-Exos directly reduced the number of macrophages (F4/80⁺) in the peritoneum.

Moreover, MSC-treated macrophages manifested as alternatively activated macrophages possess anti-inflammatory properties [42]. KEGG analysis indicated that exosome treatment involved many inflammatory pathways and immune-related pathways. Therefore, BMSC-Exos might regulate various proinflammatory signaling pathways, inhibit the activation of immune cells such as macrophages, and alleviate peritoneal inflammation.

Myofibroblasts also emerged to be the principal effector population accounting for peritoneal fibrosis [43]. In a rat model of peritoneal fibrosis, MSCs inhibited myofibroblast accumulation in the peritoneum [9]. Similarly, BMSC-derived exosomes significantly inhibited α-SMA expression, a myofibroblast-specific marker, in the peritoneum. Myofibroblasts play crucial roles in fibrosis progression, so elucidating their origin has scientific and practical implications. Lineage tracing revealed fibroblasts as major precursor cells of myofibroblasts in peritoneal fibrosis, playing a major role in promoting peritoneal thickening and fibrosis [44]. BMSC-derived exosomes penetrated deeply into the submesothelial fibroblasts (collagen-I+ vimentin+) in the peritoneum (Fig. 1G), suggesting that exosomes might regulate some biological processes of submesothelial fibroblasts to alleviate peritoneal fibrosis. MSC-Exos could regulate cell differentiation (Figs. 4D, E, 5C and Supplementary Table S3), which might involve fibroblast-to-myofibroblast transdifferentiation. However, the specific mechanism of exosomes regulation remains unclear.

Exosomes, which deliver multiple cargoes (eg, proteins, lipids, miRNAs, and mRNAs) from donor to recipient cells, can mediate cell processes such as inflammatory response, cell differentiation, cell proliferation, and cell metabolism [45]. However, the therapeutic effect of exosomes depends on the miRNAs in exosomes. For instance, MSC-Exos-mediated miR-181-5p transfer activated autophagy and inhibited liver fibrosis [46]. MSC-Exos-derived miR-199a-3p inhibited heart fibrosis by initiating autophagy [17]. Exosomes from miR-133a-modified BMSCs ameliorated fibrosis [47]. Comprehensively exploring miRNA expression in exosomes from different MSCs is necessary to offer more strategies and potential therapeutic targets when managing fibrotic disorders. Therefore, by classifying the signaling pathways and biological roles that miRNA may affect, we found that the peritoneal protection provided by BMSC-Exos may be completed by a series of miRNAs of different functions. However, further research is needed.

Conclusions

Our study provides the first comprehensive insight into the therapeutic effects of exosomes on PD-associated peritoneal fibrosis. BMSC-Exos treatment exerts a widespread protective effect mainly on peritoneal inflammation and fibrosis through a series of miRNAs. Therefore, BMSC-Exos therapy might be a promising cell-free treatment modality for PD-associated peritoneal fibrosis. The safety and efficacy of BMSC-Exos treatment for clinical translation need to be further examined.

Data Availability

All data are available from the corresponding authors on reasonable request.

Footnotes

Author Disclosure Statement

The authors have no conflicts of interest to declare.

Funding Information

This work was supported by the grants from the National Natural Science Foundation of China (82200838, 82270768) and Chongqing Technology Innovation Project (2022YSZX-JCX0007CSTB, 2023QNXM007, and SKLKF202202).

Supplementary Material

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Figure S1

Supplementary Figure S2

References

Howell

, Walker

, and Howard

. (2019). Cost effectiveness of dialysis modalities: a systematic review of economic evaluations. Appl Health Econ Health Policy, 17:315–330.

Cho

and Johnson

. (2014). Peritoneal dialysis-related peritonitis: towards improving evidence, practices, and outcomes. Am J Kidney Dis, 64:278–289.

Javaid

, Khan

, and Subramanian

. (2019). Peritoneal dialysis as initial dialysis modality: a viable option for late-presenting end-stage renal disease. J Nephrol, 32:51–56.

Yeates

, Zhu

, Vonesh

, Trpeski

, Blake

, and Fenton

. (2012). Hemodialysis and peritoneal dialysis are associated with similar outcomes for end-stage renal disease treatment in Canada. Nephrol Dial Transplant, 27:3568–3575.

Zhou

, Bajo

, Del Peso

, Yu

, and Selgas

. (2016). Preventing peritoneal membrane fibrosis in peritoneal dialysis patients. Kidney Int, 90:515–524.

Zhang

, Jiang

, and Ni

. (2017). Strategies for preventing peritoneal fibrosis in peritoneal dialysis patients: new insights based on peritoneal inflammation and angiogenesis. Front Med, 11:349–358.

Bajo

, Del Peso

, and Teitelbaum

. (2017). Peritoneal membrane preservation. Semin Nephrol, 37:77–92.

Ueno

, Nakashima

, Doi

, Kawamoto

, Honda

, Yokoyama

, Doi

, Higashi

, Yorioka

, et al. (2013). Mesenchymal stem cells ameliorate experimental peritoneal fibrosis by suppressing inflammation and inhibiting TGF-beta1 signaling. Kidney Int, 84:297–307.

Nagasaki

, Nakashima

, Tamura

, Ishiuchi

, Honda

, Ueno

, Doi

, Kato

, and Masaki

(2021). Mesenchymal stem cells cultured in serum-free medium ameliorate experimental peritoneal fibrosis. Stem Cell Res Ther, 12:203.

10.

Patel

, Shah

, and Srivastava

. (2013). Therapeutic potential of mesenchymal stem cells in regenerative medicine. Stem Cells Int, 2013:496218.

11.

Chen

, Wong

, and Gurtner

. (2012). Therapeutic potential of bone marrow-derived mesenchymal stem cells for cutaneous wound healing. Front Immunol, 3:192.

12.

Nagaishi

, Mizue

, Chikenji

, Otani

, Nakano

, Konari

, and Fujimiya

. (2016). Mesenchymal stem cell therapy ameliorates diabetic nephropathy via the paracrine effect of renal trophic factors including exosomes. Sci Rep, 6:34842.

13.

, Li

, Du

, Zhang

, Li

, Chu

, Han

, Galons

, Zhang

, Sun

, and Yu

. (2020). Exosomes from different cells: characteristics, modifications, and therapeutic applications. Eur J Med Chem, 207:112784.

14.

Murray LMA and Krasnodembskaya

. (2019). Concise Review: intercellular communication via organelle transfer in the biology and therapeutic applications of stem cells. Stem Cells, 37:14–25.

15.

Sun

, Liu

, Zhang

, Cao

, Liu

, and Li

. (2021). Mesenchymal stem cells-derived exosomes for drug delivery. Stem Cell Res Ther, 12:561.

16.

Alasmari

, Abdelfattah-Hassan

, El-Ghazali

, Abdo

, Ibrahim

, ElSawy

, El-Shetry

, Saleh

, Abourehab

MAS

, and Mahfouz

. (2022). Exosomes derived from BM-MSCs mitigate the development of chronic kidney damage post-menopause via interfering with fibrosis and apoptosis. Biomolecules, 12:663.

17.

Fan

, Wang

, Chen

, Ye

, and Fan

. (2022). Exosomes derived from bone mesenchymal stem cells attenuate myocardial fibrosis both in vivo and in vitro via autophagy activation: the key role of miR-199a-3p/mTOR pathway. Hum Cell, 35:817–835.

18.

, Li

, Chen

, Wang

, Zhao

, He

, Zhang

, and Jiang

. (2022). hMSCs-derived exosome circCDK13 inhibits liver fibrosis by regulating the expression of MFGE8 through miR-17-5p/KAT2B. Cell Biol Toxicol [Epub ahead of print]; DOI: 10.1007/s10565-022-09714-4.

19.

Sun

, Shi

, Duan

, Zhang

, and Wang

. (2022). Exosomal microRNA-618 derived from mesenchymal stem cells attenuate the progression of hepatic fibrosis by targeting Smad4. Bioengineered, 13:5915–5927.

20.

, Fan

, Wu

, Zhang

, Chu

, Wang

, and Zhuang

. (2022). Exosomes from bone marrow mesenchymal stem cells with overexpressed Nrf2 inhibit cardiac fibrosis in rats with atrial fibrillation. Cardiovasc Ther, 2022:2687807.

21.

, McLoughlin

, Brodovitch

, Moulin

, Brouckaert

, Casadei

, Feron

, Topley

, Balligand

, and Devuyst

. (2010). Nitric oxide synthase isoforms play distinct roles during acute peritonitis. Nephrol Dial Transplant, 25:86–96.

22.

Pertea

, Pertea

, Antonescu

, Chang

, Mendell

, and Salzberg

. (2015). StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol, 33:290–295.

23.

Robinson

, McCarthy

, and Smyth

. (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics, 26:139–140.

24.

Yokoi

, Kasahara

, Mori

, Ogawa

, Kuwabara

, Imamaki

, Kawanishi

, Koga

, Ishii

, et al. (2012). Pleiotrophin triggers inflammation and increased peritoneal permeability leading to peritoneal fibrosis. Kidney Int, 81:160–169.

25.

Velloso

, Otoni

, de Paula Sabino

, de Castro

, Pinto

, Marinho

, and Rios

. (2014). Peritoneal dialysis and inflammation. Clin Chim Acta, 430:109–114.

26.

Ebrahim

, Ahmed

, Hussien

, Dessouky

, Farid

, Elshazly

, Mostafa

, Gazzar

WBE

, Sorour

, et al. (2018). Mesenchymal stem cell-derived exosomes ameliorated diabetic nephropathy by autophagy induction through the mTOR signaling pathway. Cells, 7:226.

27.

Nassar

, El-Ansary

, Sabry

, Mostafa

, Fayad

, Kotb

, Temraz

, Saad

, Essa

, and H

Adel

. (2017). Erratum to: umbilical cord mesenchymal stem cells derived extracellular vesicles can safely ameliorate the progression of chronic kidney diseases. Biomater Res, 21:3.

28.

Herrera

, Henke

, and Bitterman

. (2018). Extracellular matrix as a driver of progressive fibrosis. J Clin Invest, 128:45–53.

29.

Shinde

, Humeres

, and Frangogiannis

. (2017). The role of alpha-smooth muscle actin in fibroblast-mediated matrix contraction and remodeling. Biochim Biophys Acta Mol Basis Dis, 1863:298–309.

30.

Stachowska-Pietka

, Poleszczuk

, Flessner

, Lindholm

, and Waniewski

. (2019). Alterations of peritoneal transport characteristics in dialysis patients with ultrafiltration failure: tissue and capillary components. Nephrol Dial Transplant, 34:864–870.

31.

Shi

, Yu

, and Sheng

. (2017). Angiogenesis and inflammation in peritoneal dialysis: the role of adipocytes. Kidney Blood Press Res, 42:209–219.

32.

, Ng

, and McIntyre

. (2017). Inflammation and peritoneal dialysis. Semin Nephrol, 37:54–65.

33.

Kang DH. (2020). Loosening of the mesothelial barrier as an early therapeutic target to preserve peritoneal function in peritoneal dialysis. Kidney Res Clin Pract, 39:136–144.

34.

Ferguson

, Wang

, Lee

, Liu

, Neelamegham

, Canty

, and Nguyen

. (2018). The microRNA regulatory landscape of MSC-derived exosomes: a systems view. Sci Rep, 8:1419.

35.

Eirin

and Lerman

. (2021). Mesenchymal stem/stromal cell-derived extracellular vesicles for chronic kidney disease: are we there yet?. Hypertension, 78:261–269.

36.

Harrell

, Jovicic

, Djonov

, Arsenijevic

, and Volarevic

. (2019). Mesenchymal stem cell-derived exosomes and other extracellular vesicles as new remedies in the therapy of inflammatory diseases. Cells, 8:1605.

37.

Kadota

, Fujita

, Araya

, Watanabe

, Fujimoto

, Kawamoto

, Minagawa

, Hara

, Ohtsuka

, et al. (2021). Human bronchial epithelial cell-derived extracellular vesicle therapy for pulmonary fibrosis via inhibition of TGF-beta-WNT crosstalk. J Extracell Vesicles, 10:e12124.

38.

Mehrotra

, Devuyst

, Davies

, and Johnson

. (2016). The current state of peritoneal dialysis. J Am Soc Nephrol, 27:3238–3252.

39.

Liao

, Andrews

, Wallace

, Khan

, Kift-Morgan

, Topley

, Fraser

, and Taylor

. (2017). Peritoneal macrophage heterogeneity is associated with different peritoneal dialysis outcomes. Kidney Int, 91:1088–1103.

40.

Yang

, Chang

, Chen

, Wu

, Yang

, and Lee

. (2021). Adipose-derived mesenchymal stem cells attenuate dialysis-induced peritoneal fibrosis by modulating macrophage polarization via interleukin-6. Stem Cell Res Ther, 12:193.

41.

Lo Sicco

, Reverberi

, Balbi

, Ulivi

, Principi

, Pascucci

, Becherini

, Bosco

, Varesio

, et al. (2017). Mesenchymal stem cell-derived extracellular vesicles as mediators of anti-inflammatory effects: endorsement of macrophage polarization. Stem Cells Transl Med, 6:1018–1028.

42.

Khosrowpour

, Hashemi

, Mohammadi-Yeganeh

, and Soudi

. (2017). Pretreatment of mesenchymal stem cells with leishmania major soluble antigens induce anti-inflammatory properties in mouse peritoneal macrophages. J Cell Biochem, 118:2764–2779.

43.

Padwal

, and Margetts

. (2016). Experimental systems to study the origin of the myofibroblast in peritoneal fibrosis. Kidney Res Clin Pract, 35:133–141.

44.

Chen

, Chang

, Pan

, Chou

, Chang

, Yeh

, Liu

, Chiang

, Chen

, et al. (2014). Lineage tracing reveals distinctive fates for mesothelial cells and submesothelial fibroblasts during peritoneal injury. J Am Soc Nephrol, 25:2847–2858.

45.

Vlassov

, Magdaleno

, Setterquist

, and Conrad

. (2012). Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta, 1820:940–948.

46.

, Zhang

, Cai

, Li

, Ma

, Xu

, and Lu

. (2017). Exosomes derived from miR-181-5p-modified adipose-derived mesenchymal stem cells prevent liver fibrosis via autophagy activation. J Cell Mol Med, 21:2491–2502.

47.

, Jin

, Ye

, Wang

, Deng

, and Zhang

. (2021). Bone marrow mesenchymal stem cell-derived exosomal MicroRNA-133a restrains myocardial fibrosis and epithelial-mesenchymal transition in viral myocarditis rats through suppressing MAML1. Nanoscale Res Lett, 16:111.

48.

, Wen

, Jiang

, Li

, Guan

, Zhang

, Deng

, Zhao

, Zheng

, et al. (2022). TNF-alpha stimulation enhances the neuroprotective effects of gingival MSCs derived exosomes in retinal ischemia-reperfusion injury via the MEG3/miR-21a-5p axis. Biomaterials, 284:121484.

49.

Liu

, Zhang

, Wang

, Dong

, Zhao

, Zhang

, Liu

, Guo

, et al. (2022). Inflammation-stimulated MSC-derived small extracellular vesicle miR-27b-3p regulates macrophages by targeting CSF-1 to promote temporomandibular joint condylar regeneration. Small, 18:e2107354.

50.

Liu

, Li

, Cao

, Zhang

, and Zhang

. (2021). Exosomal miR-132-3p from mesenchymal stem cells alleviated LPS-induced acute lung injury by repressing TRAF6. Autoimmunity, 54:493–503.

51.

Fang

, Zhang

, Wang

, He

, Liu

, Meng

, Peng

, Xu

, Fan

, et al. (2020). Small extracellular vesicles derived from human mesenchymal stromal cells prevent group 2 innate lymphoid cell-dominant allergic airway inflammation through delivery of miR-146a-5p. J Extracell Vesicles, 9:1723260.

52.

, Chen

, Hu

, Wu

, Li

, Gong

, Lin

, Wang

, et al. (2019). Attenuation of experimental autoimmune hepatitis in mice with bone mesenchymal stem cell-derived exosomes carrying MicroRNA-223-3p. Mol Cells, 42:906–918.

53.

Qiu

, Liu

, and Fu

. (2021). MiR-129-5p shuttled by human synovial mesenchymal stem cell-derived exosomes relieves IL-1beta induced osteoarthritis via targeting HMGB1. Life Sci, 269:118987.

54.

Zheng

, Liu

, Chen

, Lin

, Luo

, Ma

, Lin

, Shen

, and Zhang

. (2021). Exosomal miR-22-3p from human umbilical cord blood-derived mesenchymal stem cells protects against lipopolysaccharid-induced acute lung injury. Life Sci, 269:119004.

55.

Zhao

, Gan

, Xu

, Hua

, and Liu

. (2020). Exosomes from MSCs overexpressing microRNA-223-3p attenuate cerebral ischemia through inhibiting microglial M1 polarization mediated inflammation. Life Sci, 260:118403.

56.

Cai

, Cai

, Zhou

, Zhuang

, Liu

, Pei

, Wang

, et al. (2021). Mesenchymal stem cell-derived exosome miR-542-3p suppresses inflammation and prevents cerebral infarction. Stem Cell Res Ther, 12:2.

57.

Wang

, Lai

, Wu

, Liu

, Wang

, and Rong

. (2021). Umbilical mesenchymal stem cell-derived exosomes facilitate spinal cord functional recovery through the miR-199a-3p/145-5p-mediated NGF/TrkA signaling pathway in rats. Stem Cell Res Ther, 12:117.

58.

Peng

, Zhao

, Peng

, Xu

, and Yu

. (2020). Exosomal miR-25-3p from mesenchymal stem cells alleviates myocardial infarction by targeting pro-apoptotic proteins and EZH2. Cell Death Dis, 11:317.

59.

Jin

, Ren

, and Qi

. (2020). Exosomal miR-9-5p secreted by bone marrow-derived mesenchymal stem cells alleviates osteoarthritis by inhibiting syndecan-1. Cell Tissue Res, 381:99–114.

60.

, Shen

, Liu

, Wang

, Zhang

, Sui

, Tang

, Du

, Yang

, et al. (2022). Bone marrow mesenchymal stem cell-derived exosomal miR-34c-5p ameliorates RIF by inhibiting the core fucosylation of multiple proteins. Mol Ther, 30:763–781.

61.

, Shen

, Jiang

, Wang

, Yang

, Mao

, Wu

, Li

, and Chen

. (2022). Mouse mesenchymal stem cell-derived exosomal miR-466f-3p reverses EMT process through inhibiting AKT/GSK3beta pathway via c-MET in radiation-induced lung injury. J Exp Clin Cancer Res, 41:128.

62.

Baral

, Uchiyama

, Yokoyama

, Sekiguchi

, Yamazaki

, Amalia

, Inoue

, Ogino

, Torii

, et al. (2021). Antifibrotic effects and mechanisms of mesenchymal stem cell-derived exosomes in a systemic sclerosis mouse model: possible contribution of miR-196b-5p. J Dermatol Sci, 104:39–47.

63.

Yan

, Cui

, and Chen

. (2022). Mesenchymal stem cell-derived exosome-loaded microRNA-129-5p Inhibits TRAF3 expression to alleviate apoptosis and oxidative stress in heart failure. Cardiovasc Toxicol, 22:631–645.

64.

Zheng

, Zhang

, Cai

, Yang

, Guo

, Li

, and Dai

. (2022). Bone marrow mesenchymal stem cell-derived exosomal microRNA-29b-3p promotes angiogenesis and ventricular remodeling in rats with myocardial infarction by targeting ADAMTS16. Cardiovasc Toxicol, 22:689–700.

65.

Zhang

, Pan

, Liu

, Li

, Tang

, Duan

, Li

, and Zhang

. (2021). Exosomes derived from human umbilical cord blood mesenchymal stem cells stimulate regenerative wound healing via transforming growth factor-beta receptor inhibition. Stem Cell Res Ther, 12:434.

66.

, Rong

, Zhou

, Zhao

, Zhuansun

, Wan

, Sun

, and Wang

. (2020). Bone marrow mesenchymal stem cells ameliorate cisplatin-induced renal fibrosis via miR-146a-5p/Tfdp2 axis in renal tubular epithelial cells. Front Immunol, 11:623693.

67.

Wang

, Gu

, Cao

, Li

, Xiang

, Hu

, and Han

. (2016). miR-877-3p targets Smad7 and is associated with myofibroblast differentiation and bleomycin-induced lung fibrosis. Sci Rep, 6:30122.

68.

Cui

, Banerjee

, Xie

, Ge

, Liu

, Matalon

, Thannickal

, and Liu

. (2016). MicroRNA-27a-3p Is a Negative Regulator of Lung Fibrosis by Targeting Myofibroblast Differentiation. Am J Respir Cell Mol Biol, 54:843–852.

69.

Liu

, Ding

, Hou

, Yu

, Nie

, and Cui

. (2021). The miR-130a-3p/TGF-betaRII axis participates in inhibiting the differentiation of fibroblasts induced by TGF-beta1. Front Pharmacol, 12:732540.

70.

, Peng

, Fang

, Wu

, and Wu

. (2022). MSCs-derived extracellular vesicles carrying miR-212-5p alleviate myocardial infarction-induced cardiac fibrosis via NLRC5/VEGF/TGF-beta1/SMAD axis. J Cardiovasc Transl Res, 15:302–316.

71.

, Wu

, Liu

, Luo

, and Wei

. (2021). Extracellular vesicles-derived miR-150-5p secreted by adipose-derived mesenchymal stem cells inhibits CXCL1 expression to attenuate hepatic fibrosis. J Cell Mol Med, 25:701–715.

72.

Xiao

, He

, Guan

, Hou

, Yan

, Xu

, Zhou

, Liu

, and Xie

. (2020). Mesenchymal stem cells reverse EMT process through blocking the activation of NF-kappaB and Hedgehog pathways in LPS-induced acute lung injury. Cell Death Dis, 11:863.

73.

Zhu

, Sun

, Zhao

, Liu

, Zhang

, Hong

, Zhu

, Lu

, et al. (2021). Macrophage migration inhibitory factor facilitates the therapeutic efficacy of mesenchymal stem cells derived exosomes in acute myocardial infarction through upregulating miR-133a-3p. J Nanobiotechnol, 19:61.

74.

, Liu

, Li

, Lu

, Jia

, and Liu

. (2020). Exosomes derived from atorvastatin-pretreated MSC accelerate diabetic wound repair by enhancing angiogenesis via AKT/eNOS pathway. Stem Cell Res Ther, 11:350.

75.

Yang

, Li

, Wang

, Xu

, Chen

, Liu

, Sun

, Li

, Gong

, et al. (2019). Exposure to blue light stimulates the proangiogenic capability of exosomes derived from human umbilical cord mesenchymal stem cells. Stem Cell Res Ther, 10:358.

76.

Chen

, Wang

, Xia

, Yan

, and Lu

. (2018). Therapeutic Potential of Mesenchymal Cell-Derived miRNA-150-5p-Expressing Exosomes in Rheumatoid Arthritis Mediated by the Modulation of MMP14 and VEGF. J Immunol, 201:2472–2482.

77.

Ning

, Li

, Yang

, Xin

, Ping

, Huang

, Gu

, and Guo

. (2021). Blocking exosomal miRNA-153-3p derived from bone marrow mesenchymal stem cells ameliorates hypoxia-induced myocardial and microvascular damage by targeting the ANGPT1-mediated VEGF/PI3k/Akt/eNOS pathway. Cell Signal, 77:109812.

78.

Yue

, Guo

, Wang

, and Jia

. (2019). Inhibition of miR-17-5p promotes mesenchymal stem cells to repair spinal cord injury. Eur Rev Med Pharmacol Sci, 23:3899–3907.

79.

Pan

, Luo

, Zhao

, Li

, and Jiang

. (2022). miR-340-5p mediates the therapeutic effect of mesenchymal stem cells on corneal neovascularization. Graefes Arch Clin Exp Ophthalmol, 260:497–507.

80.

Gao

, Sun

, Gong

, Wang

, and Hou

. (2016). MicroRNA-195a-3p inhibits angiogenesis by targeting Mmp2 in murine mesenchymal stem cells. Mol Reprod Dev, 83:413–423.

81.

Davis

, Dukes

, Drewry

, Helwa

, Johnson

, Isales

, Hill

, Liu

, Shi

, Fulzele

, and Hamrick

. (2017). MicroRNA-183-5p Increases with Age in bone-derived extracellular vesicles, suppresses bone marrow stromal (Stem) cell proliferation, and induces stem cell senescence. Tissue Eng Part A, 23:1231–1240.

82.

Feng

, Jin

, Ming-Yu

, Yang

, Xu

, You-Xing

, Xu-Ting

, Wan

, Yun-Jiao

, et al. (2021). MiR-6924-5p-rich exosomes derived from genetically modified Scleraxis-overexpressing PDGFRalpha(+) BMMSCs as novel nanotherapeutics for treating osteolysis during tendon-bone healing and improving healing strength. Biomaterials, 279:121242.

83.

Gao

, Yang

, Huang

, Wang

, and Li

. (2018). miR-135a-5p affects adipogenic differentiation of human adipose-derived mesenchymal stem cells by promoting the Hippo signaling pathway. Int J Clin Exp Pathol, 11:1347–1355.

84.

Yao

, Mao

, Wu

, Zhu

, Lu

, Huang

, Guo

, Wang

, Zhu

, Li

, and Lu

. (2021). Exosomal circ_0030167 derived from BM-MSCs inhibits the invasion, migration, proliferation and stemness of pancreatic cancer cells by sponging miR-338-5p and targeting the Wif1/Wnt8/beta-catenin axis. Cancer Lett, 512:38–50.

85.

Zhang

, Zhu

, Zhang

, Liu

, Sun

, Li

, Na

, Xian

, Wang

, and Teng

. (2018). miR-542-3p prevents ovariectomy-induced osteoporosis in rats via targeting SFRP1. J Cell Physiol, 233:6798–6806.

86.

Bork

, Horn

, Castoldi

, Hellwig

, Ho

, and Wagner

. (2011). Adipogenic differentiation of human mesenchymal stromal cells is down-regulated by microRNA-369-5p and up-regulated by microRNA-371. J Cell Physiol, 226:2226–2234.

87.

Yang

, Luo

, Chen

, You

, and Chen

. (2021). MicroRNAs as important regulators mediate the multiple differentiation of mesenchymal stromal cells. Front Cell Dev Biol, 9:619842.

88.

Mao

, Zhang

, Hu

, Zhang

, Chang

, Huang

, Liao

, and Kang

. (2018). Exosomes derived from miR-92a-3p-overexpressing human mesenchymal stem cells enhance chondrogenesis and suppress cartilage degradation via targeting WNT5A. Stem Cell Res Ther, 9:247.

89.

Lin

, Wu

, Zhang

, Yang

, Guan

, Hou

, and Wu

. (2014). MiR-335-5p promotes chondrogenesis in mouse mesenchymal stem cells and is regulated through two positive feedback loops. J Bone Miner Res, 29:1575–1585.

90.

Zhao

, Gu

, Wang

, Qin

, Wang

, Huang

, Zhang

, Qu

, Zhang

, et al. (2021). miR-129-5p Promotes Osteogenic Differentiation of BMSCs and Bone Regeneration via Repressing Dkk3. Stem Cells Int, 2021:7435605.

91.

Costa

, Raimondi

, Conigliaro

, Salamanna

, Carina

, De Luca

, Bellavia

, Alessandro

, Fini

, and Giavaresi

. (2017). Hypoxia-inducible factor 1Alpha may regulate the commitment of mesenchymal stromal cells toward angio-osteogenesis by mirna-675-5P. Cytotherapy, 19:1412–1425.

92.

Long

, Cen

, Zhong

, Zhou

, and Zhong

. (2021). FOXO3 is targeted by miR-223-3p and promotes osteogenic differentiation of bone marrow mesenchymal stem cells by enhancing autophagy. Hum Cell, 34:14–27.

93.

Zhang

, Yuan

, Sun

, Zhou

, and Yan

. (2020). miR-199b-5p promoted chondrogenic differentiation of C3H10T1/2 cells by regulating JAG1. J Tissue Eng Regen Med, 14:1618–1629.

94.

Hwang

, Park

, Lee

, Kim

, Lee

, Choi

, You

, Kim

, and Suh

. (2014). miR-140-5p suppresses BMP2-mediated osteogenesis in undifferentiated human mesenchymal stem cells. FEBS Lett, 588:2957–2963.

95.

Chen

, Yang

, Song

, Li

, Liu

, and Wu

. (2021). MicroRNA-409-3p promotes osteoblastic differentiation via activation of Wnt/beta-catenin signaling pathway by targeting SCAI. Biosci Rep, 41:BSR20201902.

96.

Guerit

, Philipot

, Chuchana

, Toupet

, Brondello

, Mathieu

, Jorgensen

, and Noel

. (2013). Sox9-regulated miRNA-574-3p inhibits chondrogenic differentiation of mesenchymal stem cells. PLoS One, 8:e62582.

97.

Lee

, Lee

, Kim

, Yeom

, Woo

, Jung

, Yun

, Park

, Han

, et al. (2021). Extracellular vesicles from adipose tissue-derived stem cells alleviate osteoporosis through osteoprotegerin and miR-21-5p. J Extracell Vesicles, 10:e12152.

98.

Wang

, Xu

, Zhao

, Xu

, Zhang

, Jiang

, Yan

, Gu

, Wu

, Wang

, and Liu

. (2016). miR-26b-3p regulates human umbilical cord-derived mesenchymal stem cell proliferation by targeting estrogen receptor. Stem Cells Dev, 25:415–426.

99.

Fan

, Deng

, Lai

, Wen

, Zeng

, Gao

, Liu

, Kong

, Zhong

, Su

, and Zhang

. (2019). Inhibition of microRNA-221-5p induces osteogenic differentiation by directly targeting smad3 in myeloma bone disease mesenchymal stem cells. Oncol Lett, 18:6536–6544.

100.

Shi

, Ma

, Yang

, Wang

, Li

, Song

, Li

, Xie

, and Dang

. (2021). miR-362-3p Targets orosomucoid 1 to promote cell proliferation, restrain cell apoptosis and thereby mitigate hypoxia/reoxygenation-induced cardiomyocytes injury. Cardiovasc Toxicol, 21:387–398.

101.

Chen

, Huang

, Lin

, Wu

, Tan

, and Chen

. (2021). MicroRNA-425-5p modulates osteoporosis by targeting annexin A2. Immun Ageing, 18:45.

102.

Jiang

, Kitaura

, Liu

, Mizoguchi

, and Liu

. (2021). The miR-155-5p inhibits osteoclast differentiation through targeting CXCR2 in orthodontic root resorption. J Periodontal Res, 56:761–773.

103.

Feng

, Ge

, Du

, Zhang

, and Liu

. (2019). MiR-34b-3p represses cell proliferation, cell cycle progression and cell apoptosis in non-small-cell lung cancer (NSCLC) by targeting CDK4. J Cell Mol Med, 23:5282–5291.

104.

Fan

, Li

, Zhu

, Dai

, Wu

, Gao

, Zhang

, and Xu

. (2021). miR-301b-3p regulates breast cancer cell proliferation, migration, and invasion by targeting NR3C2. J Oncol, 2021:8810517.

105.

Huang

, Chen

, Fan

, and Yu

. (2022). Exosomal microRNA-140-3p from human umbilical cord mesenchymal stem cells attenuates joint injury of rats with rheumatoid arthritis by silencing SGK1. Mol Med, 28:36.

106.

Cao

, Wang

, Tang

, Zhao

, Guo

, Yue

, Zhang

, Liu

, et al. (2021). Circulating exosomes repair endothelial cell damage by delivering miR-193a-5p. J Cell Mol Med, 25:2176–2189.

107.

, Jin

, Cui

, and Xie

. (2021). Human umbilical cord mesenchymal stem cells-derived exosomal microRNA-17-3p ameliorates inflammatory reaction and antioxidant injury of mice with diabetic retinopathy via targeting STAT1. Int Immunopharmacol, 90:107010.

108.

Zhang

, Wei

, Liu

, Zhang

, Wang

, Zhou

, Zou

, Fan

, Chi

, Sun

, and Wang

. (2021). Exosomal microRNA-98-5p from hypoxic bone marrow mesenchymal stem cells inhibits myocardial ischemia-reperfusion injury by reducing TLR4 and activating the PI3K/Akt signaling pathway. Int Immunopharmacol, 101:107592.

109.

Liang

, Qin

, Luo

, Yin

, Shi

, Wei

, and Ma

. (2022). Exosomal microRNA-133b-3p from bone marrow mesenchymal stem cells inhibits angiogenesis and oxidative stress via FBN1 repression in diabetic retinopathy. Gene Ther, 29:710–719.

110.

, Wang

, Cai

, Zhou

, Li

, and Fu

. (2021). Exosomes from human umbilical cord mesenchymal stem cells inhibit ROS production and cell apoptosis in human articular chondrocytes via the miR-100-5p/NOX4 axis. Cell Biol Int, 45:2096–2106.

111.

Liu

, Yu

, Zhou

, Wang

, Sun

, Chen

, and Hou

. (2020). Lipopolysaccharide-stimulated bone marrow mesenchymal stem cells-derived exosomes inhibit H2O2-induced cardiomyocyte inflammation and oxidative stress via regulating miR-181a-5p/ATF2 axis. Eur Rev Med Pharmacol Sci, 24:10069–10077.

112.

Liu

, Lin

, Qiao

, and Zhang

. (2022). Exosomes derived from lncRNA TCTN2-modified mesenchymal stem cells improve spinal cord injury by miR-329-3p/IGF1R Axis. J Mol Neurosci, 72:482–495.

113.

, Liu

, Li

, and He

. (2021). miR-125a-5p regulates osteogenic differentiation of human adipose-derived mesenchymal stem cells under oxidative stress. Biomed Res Int, 2021:6684709.

114.

Mao

, Zhao

, Zhang

, and Zhao

. (2022). MiR-183-5p overexpression in bone mesenchymal stem cell-derived exosomes protects against myocardial ischemia/reperfusion injury by targeting FOXO1. Immunobiology, 227:152204.

115.

Zhang

, Bai

, Yi

, Hu

, and Hao

. (2021). Overexpression of miR-338-5p in exosomes derived from mesenchymal stromal cells provides neuroprotective effects by the Cnr1/Rap1/Akt pathway after spinal cord injury in rats. Neurosci Lett, 761:136124.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.02 MB

1.69 MB

0.31 MB