The Human Amnion Epithelial Cell Secretome Decreases Hepatic Fibrosis in Mice with Chronic Liver Fibrosis

Background: Hepatic stellate cells (HSCs) are the primary collagen-secreting cells in the liver. While HSCs are the major cell type involved in the pathogenesis of liver fibrosis, hepatic macrophages also play an important role in mediating fibrogenesis and fibrosis resolution. Previously, we observed a reduction in HSC activation, proliferation, and collagen synthesis following exposure to human amnion epithelial cells (hAEC) and hAEC-conditioned media (hAEC-CM). This suggested that specific factors secreted by hAEC might be effective in ameliorating liver fibrosis. hAEC-derived extracellular vesicles (hAEC-EVs), which are nanosized (40–100 nm) membrane bound vesicles, may act as novel cell–cell communicators. Accordingly, we evaluated the efficacy of hAEC-EV in modulating liver fibrosis in a mouse model of chronic liver fibrosis and in human HSC. Methods: The hAEC-EVs were isolated and characterized. C57BL/6 mice with CCl4-induced liver fibrosis were administered hAEC-EV, hAEC-CM, or hAEC-EV depleted medium (hAEC-EVDM). LX2 cells, a human HSC line, and bone marrow-derived mouse macrophages were exposed to hAEC-EV, hAEC-CM, and hAEC-EVDM. Mass spectrometry was used to examine the proteome profile of each preparation. Results: The extent of liver fibrosis and number of activated HSCs were reduced significantly in CCl4-treated mice given hAEC-EVs, hAEC-CM, and hAEC EVDM compared to untreated controls. Hepatic macrophages were significantly decreased in all treatment groups, where a predominant M2 phenotype was observed. Human HSCs cultured with hAEC-EV and hAEC-CM displayed a significant reduction in collagen synthesis and hAEC-EV, hAEC-CM, and hAEC-EVDM altered macrophage polarization in bone marrow-derived mouse macrophages. Proteome analysis showed that 164 proteins were unique to hAEC-EV in comparison to hAEC-CM and hAEC-EVDM, and 51 proteins were co-identified components with the hAEC-EV fraction. Conclusion: This study provides novel data showing that hAEC-derived EVs significantly reduced liver fibrosis and macrophage infiltration to an extent similar to hAEC-EVDM and hAEC-CM. hAEC-EV-based therapy may be a potential therapeutic option for liver fibrosis.


INTRODUCTION
Hepatic fibrosis results from chronic inflammatory liver injury attributed to many factors including steatohepatitis, viral hepatitis, alcohol, toxins, and autoimmune disease. Chronic liver injury may result in a dysregulated wound-healing response with persistent infiltration of inflammatory cells and accumulation of extracellular matrix (ECM) due to pathogenic activation of quiescent hepatic stellate cells (HSCs) with subsequent phenotypic transformation into collagen-secreting myofibroblasts. If this response is persistent, normal hepatic architecture is altered by extensive fibrosis and loss of functional hepatocyte mass leading to cirrhosis and portal hypertension. Patients with cirrhosis are at increased risk of liver failure and hepatocellular carcinoma (HCC) (Liedtke et al., 2013). Currently, the only effective therapy for patients with end-stage liver disease is liver transplantation, a complex surgical procedure reliant on donor availability (Francoz et al., 2007). The complexity of the procedure, an increase in patients requiring liver transplantation and donor shortages, demonstrates the urgent need for an alternate therapy.
Stem cells have been investigated as a potential therapy to treat end-stage liver disease. Mesenchymal stromal cells (MSC) have shown encouraging preclinical results in animal models of liver fibrosis (Chang et al., 2009;Cho et al., 2011;Huang et al., 2013) and in human clinical trials (Amin et al., 2013;Mohamadnejad et al., 2013). However, there are several arguments against the clinical use of MSC, including the possibility of malignant transformation and the requirement for in vitro expansion prior to clinical use (Baglio et al., 2012).
The human placenta contains several types of stem and stem-like cells including human amnion epithelial cells (hAEC). hAEC are non-tumorigenic and due to their low expression of HLA-Class IA and absent expression of Class II molecules do not induce host immune rejection (Wolbank et al., 2007;Pratama et al., 2011). hAEC are highly abundant -approximately 150 × 10 6 cells can be isolated from a single-term amniotic membrane, which is adequate for clinical use without the need for in vitro expansion (Murphy et al., 2010). We and others have shown the anti-fibrotic effect of hAEC in mouse models of bleomycin-induced lung fibrosis (Murphy et al., 2011), and in both acute (Manuelpillai et al., 2010) and chronic (Manuelpillai et al., 2012) liver fibrosis induced by carbon tetrachloride (CCl 4 ). Moreover, studies have shown that hAEC have low rates of engraftment in injured tissue (Vosdoganes et al., 2011(Vosdoganes et al., , 2013, which suggests that hAEC mediate their effects through release of paracrine factors. We have shown that hAEC-conditioned media (hAEC-CM) contains soluble factors which suppress proliferation, activation, and collagen production and induce apoptosis of human HSC (Hodge et al., 2014).
Evidence that stem cell conditioned media alone could exert therapeutic effects has given rise to a new theory on the mechanisms of certain cell therapies. For example, the MSC secretome is thought to be responsible for many of its physiological functions (Makridakis et al., 2013;Lee S.K. et al., 2015). More recently, crucial evidence has come to light suggesting that extracellular vesicles (EVs) are the effectors of MSC paracrine actions (Baglio et al., 2012). EVs are complex membrane enclosed nanoparticles that carry a cargo of select proteins, RNAs, and lipids (Xu et al., 2016). They are categorized by their size and biogenesis and include microvesicles (100-1000 nm), apoptotic vesicles (20-1500 nm), ectosomes (50-200 nm), and exosomes (40-150 nm) (Xu et al., 2016). However, EVs derived from hAEC (hAEC-EV) have yet to be characterized and as such their ability to reduce fibrosis following transplantation remains unknown. In this study, we isolated hAEC-EV by serial ultracentrifugation, performed in-depth characterization of isolated EVs and their protein cargo, and investigated the therapeutic efficacy of hAEC-EV in reducing hepatic fibrosis.

Isolation of hAEC
Human amnion epithelial cells (hAEC) were isolated from the placentas of 16 healthy women undergoing cesarean section at term (37-40 weeks gestation) as described previously (Murphy et al., 2010;Xu et al., 2016). Written informed consent was obtained from each woman. The study was approved by the Monash Health Human Research Ethics Committee (approval number: 01067B).

hAEC-Conditioned Media (CM)
The hAEC-CM was prepared by culturing 10 million hAEC in chemically defined, serum-free Ultraculture medium (Lonza, Walkersville, MD, United States). Cultures were maintained for 4 days at 37 • C in a humidified chamber containing 5% CO 2 prior to harvesting conditioned media.

Isolation of hAEC-EV and Extracellular Vesicle Depleted Media (EVDM)
Serial centrifugation was used to obtain EV and EVDM from collected hAEC-CM (Ng et al., 2013). Briefly, hAEC-CM was centrifuged at 300 g for 10 min at 4 • C and at 2000 g for 10 min at 4 • C to remove cells and cellular debris. The supernatant then was collected and centrifuged at 10,000 g for 30 min at 4 • C to remove large shed microvesicles (Meldolesi, 2015). The supernatant was subjected to ultracentrifugation 110,000 g for 90 min at 4 • C (KQ424, Optima L-90K Ultracentrifuge, Beckman, Australia). The supernatant (EV-depleted media, EVDM) was collected and the pellet was washed by resuspending in PBS and underwent a final ultracentrifugation step at 110,000 g for 90 min at 4 • C. The washed EV pellet was then resuspended in PBS and stored in aliquots. All hAEC components, EV, CM, and EVDM, were stored at −80 • C until required.

Transmission Electron Microscopy
Extracellular vesicles (EVs) suspended in PBS were placed on a formvar-carbon-coated electron microscope grid for 20 min then fixed in 1% glutaraldehyde for 5 min. Grids were then placed in uranyl-oxalate solution (UOA) followed by methylose-cellulose. These were then thoroughly dried before being subjected to a scanning electron microscope (H7500, Hitachi, Japan) at 70 kV.

Nanoparticle Tracking Analysis
The diameter and concentration of vesicles were determined using a NanoSight NS300 system (NanoSight technology, Malvern, United Kingdom) equipped with a blue laser (488 nm). Briefly, EVs and EVDM loaded into a flow-cell top plate using a syringe pump. Three videos (1 min) were recorded for each sample, merged and analyzed by NTA software (Build 3.1.45).

Animals
A male C57Bl/6J mice of 6-to 8-weeks-old were purchased from Monash Animal Services, Melbourne, VIC, Australia and maintained in pathogen-free conditions at the Monash Medical Centre Animal Facility. Twelve hourly dark-light cycles were maintained with food and water access provided ad libitum. The Monash University Animal Ethics Committee approved all animal experiments and mice received care under the Australian Code of Practice for the care and use of animals for scientific purposes.

CCl 4 , CM, EVDM, and EV Administration
Mice were divided into 5 groups (n = 6-8) and, other than the healthy untreated group, each group received intraperitoneal injections (IP) of carbon tetrachloride (CCl 4 ) twice weekly for 12 weeks at 1 µL/g body weight, diluted 1:10 in olive oil as previously described (Manuelpillai et al., 2012). Eight weeks later, when bridging fibrosis was evident, mice were administered three intravenous doses of either 350 µL CM, 350 µL EVDM (∼2 × 10 6 particles), or 1 µg EV (∼24 × 10 6 particles) in 350 µL saline or saline only (as controls) weekly for the last 4 weeks. All mice were culled at week 12 after commencing CCl 4 administration and blood and liver tissue were collected.

Immunohistochemistry
Four-micron-thick paraffin sections of liver tissue from untreated and treated mice as described above were dewaxed and rehydrated and heat-mediated antigen retrieval performed by incubation with 10 mM sodium citrate (pH 6). Sections were then incubated with 0.3% (v/v) H 2 O 2 for 15 min and blocked with a universal protein blocking solution for 1 h. Primary antibodies F4/80 (MCA497, Bio-Rad, Puchheim, Germany, 1:600) and α-Smooth Muscle Actin (α-SMA) (A5228-200UL, Sigma-Aldrich, St. Louis, MO, United States, 1:1500) were applied and the tissue sections were incubated in a humidified chamber overnight at 4 • C or 30 min at room temperature, respectively. The sections were then washed and biotinylated rabbit anti-mouse IgG2a (E0464, Dako, Carpinteria, CA, United States, 1:500,) and rabbit anti-rat IgG (E0468, Dako, Carpinteria, CA, United States, 1:150) were applied for 1 h at room temperature followed by visualization using the Vectastain ABC HRP kit (Vector Laboratories, Burlingame, CA, United States) and DAB substrate (Dako, Carpinteria, CA, United States).

Collagen Synthesis
Hepatic stellate cell (HSC) collagen synthesis was analyzed as described earlier (Hodge et al., 2014). Briefly, human immortalized HSCs (LX2 cell line, a kind gift of Professor Scott Friedman, NY, United States) were serum starved in DMEM containing 5% FBS followed by culture in Ultraculture media overnight at 37 • C. In the treatment groups, cells were cultured in 50% Ultraculture media and 50% hAEC-CM, 50% hAEC-EVDM, or 50% PBS with 10 µg EV. In the control groups, HSCs were cultured either in 100% Ultraculture media as a control for CM and EVDM or 50% Ultraculture media and 50% PBS as a control for EV. [ 3 H] Proline (1 µCi, PerkinElmer, Boston, MA, United States) was added to each sample.

Image Quantification and Analysis
Sirius red, F4/80, and α-SMA immunostaining were quantified in five non-overlapping fields of view per animal using a Olympus BX41 upright microscope at 10× magnification. A mean of means was calculated for each experimental group using the threshold function in the ImageJ software package (NIH Image, Bethesda, MD, United States). Data are represented as percentage (%) of positive area per field. M1 and M2 macrophages were identified as F4/80 + /CD86 + and F4/80 + /CD206 + , respectively, in five non-overlapping fields of view and normalized to the number of DAPI + cells using an Olympus FV1200 confocal microscope at 10× magnification. We carried out negative controls in the absence of primary antibodies for all stains to indicate the level of background.
The mass spectrometer (MS) was operated in data-dependent mode where the top 10 most abundant precursor ions in the survey scan (350-1500 Th) were selected for MS/MS fragmentation. Survey scans were acquired at a resolution of 60,000, with MS/MS resolution of 15,000. Unassigned precursor ion charge states and singly charged species were rejected, and peptide match disabled. The isolation window was set to 1.4 Th and selected precursors fragmented by HCD with normalized collision energies of 25 with a maximum ion injection time of 110 ms. Ion target values were set to 3e6 and 1e5 for survey and MS/MS scans, respectively. Dynamic exclusion was activated for 30 s. Data were acquired using Xcalibur software v4.0 (Thermo Fisher Scientific).

Database Searching and Protein Identification
Raw data were preprocessed as described (Gorshkov et al., 2015) and processed using MaxQuant (Cox and Mann, 2008) (v1.5.8.3) with Andromeda (v1.5.6) using a Human-only (UniProt #133,798 entries) sequence database (March 2017). Data were searched as described (Gopal et al., 2015;Greening et al., 2016b) with a parent tolerance of 10 ppm, fragment tolerance of 0.5 Da, and minimum peptide length 7, with false discovery rate 1% at the peptide and protein levels, with peptide lists generated from a tryptic digestion with up to two missed cleavages, cysteine carbamidomethylation as fixed modification, and methionine oxidation and protein N-terminal acetylation as variable modifications (Luber et al., 2010). Contaminants and reverse identification were excluded from further data analysis. For pathway analyses, Kyoto Encyclopedia of Genes and Genomes (KEGG) and NIH Database for Annotation, Visualization and Integrated Discovery Bioinformatics Resources 6.7 (DAVID) resources were utilized using recommended analytical parameters (Huang da et al., 2009). For gene ontology enrichment and network analyses, UniProt 1 database resource (biological process, molecular function) was utilized.

Statistics
Data were analyzed using GraphPad Prism version 6.0 software for Mac OSX (GraphPad Software, San Diego, CA, United States). Multiple comparisons between different groups were analyzed by one-way ANOVA with post hoc Bonferroni correction. An unpaired t-test was performed to compare between control and EV in the in vitro collagen synthesis experiment. Data are shown as mean ± SEM. Differences were considered significant at P < 0.05.

Characterization of hAEC-EV
Extracellular vesicles released by hAEC (hAEC-EV) were prepared using serial ultracentrifugation as described previously (Ng et al., 2013). Western blot analysis showed that hAEC-EV expressed specific exosome markers including Alix, CD81, and CD63, which were absent in hAEC-EVDM ( Figure 1A). Transmission electron microscopy showed that hAEC-EV displayed cup-shaped morphology and had a size of approximately 40-100 nm ( Figure 1B). Nanoparticle tracking analysis was used to determine size distribution of EVs, which displayed a mean 133.1 nm diameter ( Figure 1C). These results showed that hAEC-EV displayed the minimal criteria of exosomes and were absent in hAEC-EVDM (Lötvall et al., 2014). However, the presence of EVs in the EVDM could not be excluded completely by nanoparticle tracking analysis (Supplementary Figure S1).

Proteomics Analysis of hAEC-EV, hAEC-CM, and hAEC-EVDM
We next compared the proteome profiles of the hAEC-EV, hAEC-CM, and hAEC-EVDM using GeLC-MS/MS (Tauro et al., 2012a,b;Greening et al., 2016aGreening et al., , 2017. For purified EVs, this resulted in 231 proteins identified (Supplementary  Table S1). We observed an abundance of typical exosome associated proteins such as tetraspanins CD9 and CD81, various Rab GTPases, and select components associated with vesicle sorting/trafficking including ARF1, LAMP1, and CLTC (Supplementary Table S1). We found 61 proteins identified in the exosome database ExoCarta (top 100 highly expressed proteins in exosomes) 2 . This supports the enrichment of select exosome marker proteins including HSPA8, CLTN, and integrins ITGA6 and ITGB1 (Figure 8A and Supplementary Table S2). In comparison with hAEC-CM ( Figure 8B) and hAEC-EVDM (Figure 8C), we found 51 components in common with the isolated EV fraction (Supplementary Table S1). Additionally, 164 proteins were unique to hAEC-EV in comparison to hAEC-CM (Supplementary Table S3) and hAEC-EVDM (Supplementary  Table S4). EV components included Milk fat globule epidermal growth factor-factor 8 (MFGE8), heat shock 72 kDa protein The percentage of HSC in the liver was significantly decreased in mice treated with CM, EVDM, and EV compared to CCl 4 only. (C) Concentration of TGF-β1 in liver was determined by ELISA and found to be significantly lower in mice treated with EV only. The data are represented as mean ± SEM. n = 6-8 per group, * P < 0.05, * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001.
When subjected to a gene ontology analysis, we observed enrichment of biological processes associated with cell-cell adhesion, receptor-mediated endocytosis, protein transport, cell surface receptor signaling pathway, integrin-mediated signaling pathway, membrane organization, and wound healing ( Figure 8D and Supplementary Table S7). Of note, we observed KEGG enrichment associated with the PI3K-Akt signaling pathway, focal adhesion, Rap1 signaling, ECM-receptor interaction, and antigen processing and presentation ( Figure 8E and Supplementary Table S8).

DISCUSSION
This study has provides the first evidence that EVs secreted by hAEC have a therapeutic potential for the treatment of liver fibrosis. Evidence of therapeutic effects of the secretome of MSC and other stem cells gave rise to a new theory, specifically that outcomes of cell therapies may be mediated by EVs (Tolar et al., 2010;Fouraschen et al., 2012;Lee et al., 2016). These vesicles, which play an important role in cell-to-cell communication, can alter the phenotype and fate of target cells (Xu et al., 2016). There is increasing evidence that exosomes influence physiological processes such as cell transformation (Greening et al., 2015a), immunoregulation (Greening et al., 2015b;Nawaz et al., 2016), and importantly, cancer progression (Atay et al., 2014;Melo FIGURE 5 | (A) Hepatic macrophages were identified by F4/80 immunohistochemistry. Mice with established fibrosis treated with CM, EVDM, and EV had significantly lower percentage of F4/80 macrophages in the liver compare to CCl 4 only. Scale bar = 200 µm, 10× magnification. (B) Quantification of liver macrophage density using ImageJ software. The data are represented as mean ± SEM. n = 6-8 per group, * P < 0.05, * * P < 0.01, * * * P < 0.001, * * * * P < 0.0001.
In this study, we investigated the therapeutic efficacy of hAEC-CM, EV, and EVDM in a mouse model of CCl 4induced chronic liver fibrosis. We found that intravenous administration of CM, EV, and EVDM derived from hAEC significantly reduced HSC number and collagen production, reduced hepatic macrophage infiltration, and polarized macrophages to a pro-reparative phenotype in vitro and in vivo. Furthermore, only hAEC-EV significantly downregulated TGF-β1 signaling.
Carbon tetrachloride-induced liver fibrosis is a wellestablished model of liver injury (Manuelpillai et al., 2012). We commenced hAEC-CM, EV, and EVDM treatments while continuing CCl 4 administration to model the persistent injury that occurs in the clinical setting and where clinical management would be relevant. During liver fibrosis, activation of HSC and subsequent transformation into myofibroblasts leads to the production of collagen and ECM accumulation. Activated HSC are widely measured by the expression of α-SMA (Lee Y.A. et al., 2015). Treatment with hAEC-CM, EV, and EVDM significantly reduced liver fibrosis, as shown by a reduction in the number of activated HSC and collagen proportionate area, even in the presence of continued CCl 4 administration. We corroborated these findings in vitro using the human HSC cell line LX2, showing that hAEC-EV directly decreased collagen production in activated HSC. Interestingly, the therapeutic effect of hAEC-EV was similar to hAEC-CM, in regard to its reduction of liver fibrosis and HSC activation, which extends our previous findings showing that hAEC-CM contained soluble factors that have anti-fibrotic effects in vitro (Hodge et al., 2014). However, EVDM had a diminished ability to reduce collagen production in vitro, which is in contrast to our in vivo results. This could be a reflection of our use of an immortalized stellate cell line, which may not reflect an in vivo effect, as such the use of primary HSC should be the subject of further investigation. Alternatively, this may indicate that EVDM exerts its anti-fibrotic effects in vivo by acting on other cell types in the liver, rather than on stellate cells themselves.
TGF-β1 is well known to activate HSCs (Pradere et al., 2013). We found that hAEC-EV dramatically reduced the protein content of TGF-β1 in the livers of CCl 4 mice; however, this was not achieved with hAEC-CM or EVDM. TGF-β was identified in both CM and EVDM and the dose in our study was 350 µl 3 times weekly for 4 weeks. A study by Huang et al. (2016) reported that MSC-CM 250 µl twice weekly for 3 weeks promoted therapeutic effects in a chronic liver fibrosis model. The higher dose in our study may explain the impaired ability of CM and EVDM to reduce hepatic TGF-β and indicates the importance of investigating the dose efficacy of CM and EVDM.
Hepatic macrophages are a heterogeneous population of cells that have a wide range of functions during homeostasis and disease (Lee Y.A. et al., 2015). Chronic liver fibrosis is associated with recruitment of macrophages that co-localize with fibrotic regions (Manuelpillai et al., 2012). Macrophage depletion using a transgenic mouse (CD11b-DTR) resulted in decreased fibrosis and HSC in chronic liver fibrosis induced by CCl 4 (Duffield et al., 2005). In the present study, we found that CCl 4 -treated mice exhibited a significant increase in F4/80 positive macrophage infiltration, which was significantly decreased by hAEC-EV exposure.
Experimental evidence suggests that macrophages exert dual functions during liver fibrosis. The activation of macrophages during the injury phase is associated with ECM accumulation and HSC activation. On the other hand, macrophages activated during recovery resulted in matrix degradation (Duffield et al., 2005;Ramachandran et al., 2012). Phenotypic polarization from classically activated macrophages (M1) to alternatively activated macrophages (M2) is dependent on signals received from the local environment (Martinez and Gordon, 2014). M1 macrophages produce high levels of pro-inflammatory cytokines and are induced by LPS and interferon-γ (IFN-γ) (Martinez and Gordon, 2014). On the other hand, M2 macrophages produce anti-inflammatory cytokines, collagen-degrading enzymes and are induced by IL-4 and IL-13 (Song et al., 2000;López-Navarrete et al., 2011). Interestingly, the mannose and scavengers receptors present in M2 macrophages are able to phagocytose ECM and apoptotic cells leading to fibrosis resolution (López-Guisa et al., 2012;Wynn and Ramalingam, 2012). In this study, we used CD86 to identify M1 macrophages and CD206 for M2 macrophages (Bility et al., 2016). However, CD206 is expressed in M2 liver macrophages as well as liver sinusoidal endothelial cells (DeLeve, 2015). We therefore identified M1 macrophages by co-localization of CD86 and F4/80 while M2 macrophages were identified by co-localization of F4/80 and CD206. We found that hAEC-EV increased liver M2 macrophages in CCl 4 mice, accompanied by a decrease in liver M1 macrophages, a similar effect seen with hAEC-CM and EVDM. This was corroborated in vitro using immortalized bone marrow macrophages. Taken together, our findings of reduced hepatic fibrosis area, reduced number of activated HSC and macrophages, reduced levels of TGF-β1 and polarization to M2 macrophages are consistent with those seen in our previous study, when we administered hAEC alone in the CCl 4 mouse model (Manuelpillai et al., 2012). Data from our current study indicate that both the vesicular fraction and whole hAEC-CM may mediate the anti-fibrotic effects observed in CCl4-induced chronic liver fibrosis.
While the field of EV research has grown exponentially in recent years, findings from our current study indicate that the soluble fraction of secreted or shed cellular products should not be entirely disregarded. The proteomic analysis of CM, EV, and EVDM indicates the presence of proteins enriched for Rap1 pathway and PI3K/Akt pathway. Rap1 is involved in the control of cell proliferation and cell adhesion (Bos et al., 2001), while PI3K/Akt is implicated in macrophage polarization, cell cycle progression, and prevention of apoptosis (Chang et al., 2003;Vergadi et al., 2017). Therefore, our data suggest the PI3K/Akt pathway may modulate macrophage polarization. Interestingly, our proteomic analysis of hAEC-EV revealed the presence of proteins that target TGF-β signaling including MFGE8, HSP72, and SOD1. MFGE8 plays a critical role in reducing pulmonary fibrosis (Atabai et al., 2009). An et al. (2017) identified MFGE8 as an anti-fibrotic factor in the umbilical cord MSC secretome that inhibits TGF-β signaling and reduces liver fibrosis in mice. Moreover, HSP72 was found to attenuate renal tubulointerstitial fibrosis in obstructive nephropathy (Mao et al., 2008) and to inhibit epithelial-to-mesenchymal transition, which promotes collagen production, via effects on Smad2 activation (Zhou et al., 2010). Finally, the anti-fibrotic potential of SOD1 on radiation-induced fibrosis is mediated by downregulation of TGF-β signaling (Emerit et al., 2006). These proteins could play a role in the reduction of collagen production, fibrosis, and TGF-β expression observed in hAEC-EV-treated mice.
In summary, our findings suggest that the hAEC secretome, comprising soluble factors in hAEC-CM, both complete and EV depleted, in addition to hAEC-EV, had beneficial effects in reducing liver fibrosis in a murine model. This is the first study to provide evidence that hAEC-derived EVs can exert a therapeutic effect similar to what has been previously reported with hAEC in an experimental model of chronic liver fibrosis. Future studies could focus on identifying the specific anti-fibrotic factors that would support development of a clinically applicable therapy.