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Systemic sclerosis or scleroderma is an autoimmune disease in which fibrosis occurs not only in the skin but also in various internal organs, such as the lungs, heart, and kidneys (Allanore
Mesenchymal stem cells derived from human tissues, such as bone marrow, adipose tissue, and placental tissue, are widely used as therapeutic agents for tissue repair and regeneration (Uccelli
Extracellular vesicles (EVs) are nano-sized (30-200 nm) vesicles that are secreted into the extracellular space (EL Andaloussi
This study investigated the effects of ASC-EVs in an
Extracellular vesicles were isolated from human adipose-derived stem cells (ASCs). Primary human ASCs were purchased from Cefobio Inc (Seoul, Korea). ASCs were cultured in high-glucose Dulbecco’s Eagle Medium (DMEM; Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and incubated at 37°C in a 5% CO2 incubator. To isolate extracellular vesicles from the media, the cells were first washed with phosphate-buffered saline (PBS), and the medium was changed to serum-free DMEM for 24 h. The conditioned medium was collected and centrifuged at 300×g for 10 min to remove any remaining cellular debris. A 0.22 μm bottle top filter was used to eliminate extra cell debris and macrovesicles in the conditioned medium. Finally, extracellular vesicles were isolated from the filtered conditioned medium using a tangential flow filtration system with a 300-kDa MWCO ultrafiltration membrane filter (OA300C12, Pall Corporation, Port Washington, NY, USA), as described previously (Jung
Extracellular vesicles were labeled and analyzed as previously described (Lee
All animal experiments were performed in accordance with the related laws and guidelines of the Institutional Animal Care and Use Committee (IACUC) of Sungkyunkwan University (SKKUIACUC2019-11-03-1). The institutional committees approved the experiments. Male, aged 6 weeks, DBA/2 mice were purchased from Orient Bio (Seongnam, Korea) and used in this study. Dermal fibrosis was induced by injecting bleomycin (9041-93-4, Cayman, Ann Arbor, MI, USA). Subcutaneous injection of bleomycin (100 μL from a stock of 0.5 mg/ml) was continued every alternate day for 6 weeks (Yamamoto
Skin tissues were harvested at the last ASC-EVs administration, fixed in formalin, and embedded in paraffin. Histological sections (5 μm) were cut from paraffin blocks and stained with H&E and Masson’s trichrome, according to a common protocol (Dees
Primary human dermal fibroblasts (HDFs) (C0135C, Thermo Fisher Scientific) were used within passages three to seven. HDFs were incubated at 37°C in a 5%CO2 incubator and cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM, DMEM-HPA; Capricorn Scientific) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. For the experiment, cells were seeded in culture dishes and incubated for 24 h. The cells were then treated with TGF-β1 (100-21, Peprotech, Cranbury, NJ, USA) or ASC-EVs for 24 h. BSA (0.1%) and PBS were used as controls for TGF-β1 and ASC-EVs, respectively.
Human dermal fibroblasts were treated with TGF-β1 and incubated at 37°C for 24 h. ASC-EVs were then added to the cells for 24 h, and 50 μL MTT (M6494, Invitrogen, Waltham, MA, USA) was then added to each well. After three hours, the MTT solution and culture media were removed and the cells were incubated with dimethyl sulfoxide for 15 min at 37°C. A plate reader measured the absorbance at a 550 nm wavelength, with a reference wavelength of 690 nm.
HDFs were washed with and collected in 1 mL of PBS. After centrifuging at 4,000 rpm for 1 min at 4°C and removal of the supernatant, the cells were resuspended in 200 μL of buffer A (40 mM Tris-Cl, 10 mM NaCl, 1 mM EDTA, 1 mM DTT, protease inhibitor, and phosphatase inhibitor) and incubated on ice for 15 min to disrupt the cell membrane. The cells were vortexed with 10% NP-40 for 10 sec and centrifuged at 12,000 rpm for 10 min at 4°C. Supernatants were separated as cytoplasmic proteins, and the nuclear protein-containing pellets were washed twice with buffer A. After washing, the pellets were resuspended in 40 μL buffer B (40 mM Tris-Cl, 420 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM DTT, protease inhibitor, and phosphatase inhibitor) and incubated on ice for 20 min. Nuclear proteins were extracted by centrifuging at 12,000 rpm for 10 min at 4°C. The supernatants were collected as nuclear proteins.
HDFs were lysed using T-PERTM (78510, Thermo Fisher Scientific). Then, 5 μg of protein were electrophoresed on 10% and 12% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TBS-T (10 mM Tris, 150 mM NaCl, and 0.1% Tween 20) for 1 h at RT and incubated overnight at 4°C with primary antibodies against α-SMA, p-SMAD2 (3108S, Cell Signaling Technology, Danvers, MA, USA), SMAD2/3 (07-408, Merck Millipore, Burlington, MA, USA), CTGF (sc-752, Santa Cruz Biotechnology, Dallas, TX, USA), lamin b (ab16048, Abcam), α-tubulin (2144S, Cell Signaling Technology), p-AKT (9271S, Cell Signaling Technology), AKT (2920S, Cell Signaling Technology), BCL2 (PC68, Merck Millipore) and GAPDH (NB300-221, Novus Biologicals, Littleton, CO, USA). After washing with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at RT. The membranes were treated with ECL solution and the labeled proteins were visualized on X-ray film (AgfaPhoto, Bayern, Germany) using a developer and fixer. ASC-EVs were lysed using 10X RIPA buffer to disrupt the lipid bilayer. ASC and ASC-EVs were detected using the same protocol as for the HDFs and incubated with primary antibodies against CD9 (ab223052, Abcam, Cambridge, UK), CD63 (ab68418, Abcam), and Alix (109201, Abcam). GM130 (12480, Cell Signaling Technology) and Calnexin (AB2301, Merck Millipore) antibodies were used as negative markers for the EVs.
Total RNA from mouse skin tissue and HDFs was extracted using RNAiso plus (9108, Takara Bio, Kusatsu, Japan) according to the manufacturer’s protocol. Total RNA was reverse-transcribed into complementary DNA using a kit (RR037A, Takara Bio). Quantitative real-time PCR was performed using TB Green (RR820A, Takara Bio). Primer sequences used for the real-time PCR are listed in Table 1 and 2. GAPDH was used as the housekeeping gene.
Table 1 Human primer list for RT-qPCR
Human gene | Sequence |
---|---|
F: ACC CAC AAT GTC CCC ATC TA R: GAA GGA ATA GCC ACG CTC AG | |
F: AGC TG ACCT GGA AGA GAA CAT T R: GCT CGG TAT GTC TTC ATG CTG | |
F: AGC CAC ATC GCT CAG ACA C R: GCC CAA TAC GAC CAA ATC C |
Table 2 Mouse primer list for RT-qPCR
Mouse gene | Sequence |
---|---|
F: ATG GCT CTG GCT CTG TAA G R: CCC ATT CCA ACC ATT ACT CC | |
F: CTG CAG ACT GGA GAA GCA GA R: GCT TGG CGA TTT TAG GTG TC | |
F: CAT GTT CAG CTT TGT GGA CCT R: GCA GCT GAC TTC AGG GAT GT | |
F: GCA ACA TGT GGA ACT CTA CCA G R: CAG CCA CTC AGG CGT ATC A | |
F: CCA GCG TT ATAA GAT CAA GAT GAC R: CTG GAC TTG TGG GCA TAT C | |
F: CAT CTC CTC TGG GAT CCA TCT R: CCA TTC TTC AGG GTG GCT AT | |
F: CAG GTG AGT GGG GCG TTA R: GCC TGC TGT TCA CAG TTG C | |
F: GCT ACC AAA CTG GAT ATA ATC AGG R: CCA GGT AGC TAT GGT ACT CCA GAA | |
F: TTG ATG GCA ACA ATC TCC AC R: CGT CCC GTA GAC AAA ATG GT |
Statistical analysis was conducted using Prism9 software (GraphPad Software Inc., Boston, MA, USA). One-way ANOVA with Tukey’s multiple comparisons test was used to compare more than three samples, while the unpaired Student’s t-test was used to assess the significance between two samples. Data are presented as mean ± standard deviation (SD). Statistical significance was determined at
To produce ASC-EVs, human adipose-derived stem cells were cultured for 3 weeks, and ASC-EVs were isolated from 1 L of conditioned medium collected during proliferation. The ASC-EVs were characterized by their shape, size, concentration, and protein markers. Cryo-TEM analysis revealed the presence of EVs with a round, spherical shape and a double-layer membrane structure (Fig. 1A). Through NTA, it was found that the mean diameter was 162 nm, which is within the typical size range of EVs (Fig. 1B). Furthermore, the concentration of EVs was determined to be 0.160 μg/μL based on microBCA protein quantification, and the particle number concentration was confirmed to be 2.17×107 particles/μL using NTA. As shown in Fig. 1C, ASC-EVs expressed EV markers, such as CD9 and Alix, whereas the negative markers GM130 and Calnexin were not observed. To investigate the surface marker proteins of ASC-EVs, anti-CD63 antibody-coated magnetic beads were incubated with ASC-EVs, which were then detected using an anti-CD81 antibody tagged with PE. The presence of CD81 in ASC-EVs was confirmed by measuring the fluorescence using confocal microscopy and FACS analysis (Fig. 1D, 1E).
An
The study investigated the effectiveness of ASC-EVs in skin fibrosis using a mouse model of bleomycin-induced fibrosis. In this model, 50 μg of bleomycin was injected subcutaneously every other day for 6 weeks, and 108 particles of ASC-EVs were subcutaneously administered three times in total at weeks 5 and 6 (Fig. 3A). After bleomycin injection, the skin thickness increased, but the administration of ASC-EVs significantly reduced this increase (Fig. 3B). Additionally, through the use of DAB staining, it was observed that the number of myofibroblasts expressing α-SMA was lower in the ASC-EVs treated group than in the group treated with NaCl for skin fibrosis (Fig. 3C). Furthermore, mRNA analysis revealed that several fibrosis markers, including
The TGF-β/SMAD signaling pathway is overactive in fibrosis and plays a major role in systemic sclerosis (Wynn, 2008). Therefore, inhibition of the TGF-β/SMAD signaling pathway is a major target for systemic sclerosis treatment (Walton
Next, the study aimed to determine the effect of ASC-EVs on the fate of myofibroblast. Myofibroblasts have the potential to either revert to fibroblasts or undergo cell death, which is typically suppressed (Hinz and Lagares, 2020). Furthermore, the AKT pathway plays a crucial role in the transformation of differentiation of cells comprising the skin tissue (Ko and Kim, 2023). Initially, it was confirmed that treatment of dermal fibroblasts with TGF- β1 at a concentration of 2.5 ng/mL for 24 h increased the levels of phosphorylated AKT, which contributes to cell proliferation, as well as BCL-2, an anti-apoptotic marker (Supplementary Fig. 2A). To confirm the effect on activated myofibroblasts, fibroblasts were stimulated with TGF- β1 for 24 h and subsequently exposed to ASC-EVs at a concentration of 108 particles/mL for 24 h. ASC-EVs treatment significantly decreased the expression levels of not only α-SMA, but also phosphorylated AKT and BCL-2 (Fig. 6A, 6B). Prior to examining the effects of ASC-EVs on cell viability, we confirmed a significant increase in cell proliferation after treatment with 2.5 ng/mL of TGF- β1 for 24 h (Supplementary Fig. 2B). To investigate whether ASC-EVs could affect cell viability, dermal fibroblasts were treated with ASC-EVs for 24 h following the activation of cell proliferation with a 24 h treatment with 2.5 ng/mL of TGF- β1. ASC-EVs treatment did not affect cell viability under normal conditions. However, under conditions where cell proliferation was enhanced by TGF- β1, ASC-EVs treatment significantly reduced cell viability (Fig. 6C). These results indicate that ASC-EVs induce cell death in a myofibroblast-specific manner.
Systemic sclerosis is an autoimmune disease characterized by inflammatory reactions and fibrosis. Although the exact cause of systemic sclerosis remains unclear, the pathogenesis of systemic sclerosis, which accumulates ECM by activation of fibroblasts via an inflammatory reaction, has been well studied. Myofibroblasts, which directly produce ECM, are the main therapeutic targets. Several groups have demonstrated that stem cell-derived extracellular vesicles can regulate fibroblast differentiation in wound-healing models (Hu
We hypothesized that ASC-EVs could alleviate fibrosis in systemic sclerosis fibrotic models, both
Both
In conclusion, we demonstrated that ASC-EVs have therapeutic effects in a bleomycin-induced mouse model and TGF-β-induced human dermal fibroblasts
This study was supported by the National Research Foundation of Korea (NRF-2019R1A2C3011422, NRF-2019R1A5A2027340). This work was also supported by the Korea Drug Development Fund (KDDF-HN21C1266) and the Ministry of Oceans and Fisheries’ R&D project, Korea (1525011845).
DGJ, JHP, and YWC are stockholders of ExoStemTech, Inc.
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