Extracellular vesicles originating from steatotic hepatocytes promote hepatic stellate cell senescence via AKT/mTOR signaling

The prevalence of metabolic dysfunction‐associated steatotic liver disease (MASLD) is increasing rapidly due to the obesity epidemic. In the inflammatory stages of MASLD (MASH), activation of hepatic stellate cells (HSCs) leads to initiation and progression of liver fibrosis. Extracellular vesicles (EVs) are released from all cell types and play an important role in intercellular communication. However, the role of EVs released from hepatocytes in the context of MASLD is largely unknown. Therefore, the present study aimed to investigate the role of EVs derived from both normal and steatotic (free fatty acid‐treated) hepatocytes on the phenotype of HSCs via the senescence pathway. Primary rat hepatocytes were treated with free fatty acids (FFAs: oleic acid and palmitic acid). EVs were collected by ultracentrifugation. EVs markers and HSCs activation and senescence markers were assessed by Western blot analysis, qPCR and cytochemistry. Reactive oxygen species (ROS) production was assessed by fluorescence assay. RNA profiles of EVs were evaluated by sequencing. We found that EVs from hepatocytes treated with FFAs (FFA‐EVs) inhibit collagen type 1 and α‐smooth muscle actin expression, increase the production of ROS and the expression of senescence markers (IL‐6, IL‐1β, p21 and senescence‐associated β‐galactosidase activity) in early activating HSCs via the AKT–mTOR pathway. Sequencing showed differentially enriched RNA species between the EVs groups. In conclusion, EVs from FFA‐treated hepatocytes inhibit HSC activation by inducing senescence via the AKT–mTOR signaling pathway. Determining the components in EVs from steatotic hepatocytes that induce HSC senescence may lead to the identification of novel targets for intervention in the treatment of MASLD in the future.


Metabolic dysfunction-associated steatotic liver disease (MASLD)
formerly known as nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease.Currently, the prevalence is 25% in the general population globally. 1MASLD is associated with obesity, the consequence of a sedentary lifestyle and excess food intake. 2 The spectrum of MASLD ranges from simple steatosis to the inflammatory stage, metabolic dysfunction-associated steatohepatitis (MASH), liver fibrosis, cirrhosis and eventually hepatocellular carcinoma.Fibrosis is the excessive deposition of extracellular matrix (ECM) in the liver and the activated hepatic stellate cell (aHSCs) is the major source of ECM. 3 Normally, hepatic stellate cells are quiescent (qHSCs) and are involved in the storage of vitamin A. In conditions of chronic inflammation, qHSCs transdifferentiate into myofibroblast-like activated HSCs.aHSCs proliferate and produce excessive amounts of ECM.It is believed that activation of HSCs involves signals from other liver cell types, such as damaged hepatocytes and/or Kupffer cells, the resident macrophages in the liver. 4 the context of MASLD, it has been suggested that steatotic hepatocytes release signals that intervene with the (early) activation of HSCs. 5 There is growing evidence showing that small extracellular vesicles (EVs) containing both exosomal (40-160 nm, average around 100 nm), and non-exosomal, for example, microvesicles (MVs) (100-1000 nm) and apoptotic bodies (50-1000 nm in diameter) 6,7 are released from various cell types and play an important role in intercellular communication. 8,9The content of EVs released from cells varies with the state of the cell: 'healthy' versus 'diseased' cells.In MASLD, EVs can be released by damaged and/or steatotic hepatocytes and these EVs can directly act on HSCs or indirectly on HSCs via Kupffer cells to promote inflammation and fibrogenesis. 10Recently, we reviewed the literature on the role of EVs in cell to cell communication in the context of MASLD. 11Several studies investigated the content of EVs and demonstrated the presence of bioactive proteins as well as various RNA species (mRNA, miRNA), also in the context of MASLD. 12miRNAs have the capacity to modulate various aspects of cellular function.Their remarkable stability positions them as promising biomarkers with potential applications in disease diagnosis and prognosis. 11,13Senescence is a state of (almost) irreversible cell cycle arrest.Induction of senescence in stellate cells has been proposed as a potential intervention to prevent and/ or reverse activation of HSCs and we have observed a reciprocal relation between the expression of activation markers and senescence markers in HSCs. 14However, it is not known whether EVs derived from steatotic hepatocytes can modulate HSCs activation and senescence.In this study, we explored the effects of EVs from hepatocytes on markers of activation and senescence in HSCs.Free fatty acid-treated hepatocytes were used as a model of steatosis, as previously described. 15Our results demonstrate that steatotic hepatocyte-derived EVs inhibit HSCs activation via the induction of senescence.

| Animals and cell isolation techniques
Specified pathogen-free male Wistar rats (180-250 g) were purchased from Charles River Laboratories Inc. Rats were housed in a 12 h light-dark cycle under standard laboratory conditions with free access to standard laboratory chow and water.All experiments were performed according to the Dutch law on welfare of laboratory animals and guidelines of the ethics committee for care and use of laboratory animals of the University of Groningen.Rats were anesthetized using isoflurane and a mixture of ketamine and medetomidine.
Hepatocytes were isolated by two-step collagenase perfusion method via the portal vein as described previously. 16Only hepatocyte isolations with a viability higher than 80% were used.Primary rat hepatocytes were plated at 1.2×10 6 cells per well in six-well plates corresponding to a confluency of 90%.The cells were cultured in William's E medium (Invitrogen) supplemented with 50 µg/mL gentamicin (Invitrogen), 100 U/mL penicillin (Lonza), 10 µg/mL streptomycin (Lonza) and 250 ng/mL fungizone (Lonza) at 37°C in an atmosphere containing 5% (v/v) CO 2 .The experiments were started after an attachment period of 4 h.Hepatic stellate cells (HSCs) were isolated from rats weighing 350-450 g.The liver was perfused via the portal vein with a buffer containing Pronase-E (Merck) and Collagenase-P (Roche).

Significance statement
The study addresses the rising prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) by exploring the role of extracellular vesicles (EVs) from hepatocytes, particularly those treated with free fatty acids (FFAs), in hepatic stellate cell (HSC) behavior.FFA-EVs inhibited fibrotic marker expression in quiescent HSCs while promoting ROS production and senescence marker expression via the AKT-mTOR pathway.RNA profiling revealed distinct EV RNA species.These findings suggest that FFA-EVs induce quiescent HSC senescence, presenting potential targets for MASLD intervention in the early stage.
Understanding the composition of FFA-treated hepatocytederived EVs provides insight into MASLD pathogenesis and therapeutic avenues.In summary, elucidating the impact of FFA-EVs on HSCs offers promise for innovative MASLD treatments.

| Treatment of cells with free fatty acids and extracellular vesicles
Primary rat hepatocytes were incubated with a mixture of free fatty acids (FFAs) consisting of oleic acid (500 µmol/L) and palmitic acid (250 µmol/L) in an aqueous solution of bovine serum albumin (BSA) as described previously. 18Hepatocytes were treated with FFAs and nontreated hepatocytes were used as a control group.After 48 h incubation, medium was collected for isolation of EVs.
Quiescent HSCs (Day 1) and activated HSCs (Day 7) were incubated in six-well plates (1.2 x 10 6 cells per well) and treated with 15 μg protein/well of EVs from hepatocytes for 3 days.After 3 days of treatment, cells were harvested for Western blot analysis, staining, ROS production and quantitative real-time polymerase chain reaction (qPCR) assays.

| Isolation of extracellular vesicles
The general term "small extracellular vesicles" including microvesicles and exosomes, will be used according to the MISEV (2018) guideline. 19In some studies, no differentiation between microvesicles and exosomes was made. 20We will use the term EVs to describe small EVs in this study.Primary rat hepatocytes were cultured in six-well plates in serum-free medium for 48 h.
EVs were isolated from conditioned medium by differential ultracentrifugation (UCF Thermo-Sorvall 80wx + ) according to a modified protocol. 21Low-speed centrifugations (2000g for 30 min and 12,000g for 45 min) were used to eliminate cell debris.The supernatants were collected and centrifuged for 70 min at 120,000g at 4°C.Pellets were washed with PBS and centrifuged again for 60 min at 100,000g at 4°C.The obtained pellets (EVs) were resuspended in PBS solution and stored in aliquots at −80°C.Protein concentrations in EVs pellets were measured using BCA protein assay kit (Pierce).

| Nanoparticle tracking analysis (NTA)
The size and concentration of EVs were assessed by nanoparticle tracking analysis (NTA) using NanoSight NS300 instrumentation (Marvel).Isolated EVs were diluted in PBS to a final concentration of 10 7 -10 8 particles/mL per count.The camera level for each sample was manually adjusted to achieve optimal visualization of particles to record videos.

| RNA isolation and quantitative RT-PCR
RNA was isolated using Tri-reagent (Sigma-Aldrich) according to the manufacturer's protocol.RNA concentrations were measured by Nano-Drop 2000c (Thermo Fisher Scientific) and 2 μg of RNA was used for reverse transcription (Sigma-Aldrich).cDNA was diluted in RNAse-free water and used for real-time polymerase chain reaction on the QuantStudio™ 3 system (Thermo Fisher Scientific).All samples were analyzed in duplicate using 36B4 as a housekeeping gene.
Relative gene expression was calculated via the 2 −ΔΔCt method.The primers and probes are listed in Table 1.

| Determination of reactive oxygen species
Intracellular reactive oxygen species (ROS) levels were measured using dihydroethidium (DHE) (Thermo Fisher Scientific).HSCs were seeded on 96-well plates.After attachment, HSCs were treated with EVs for 72 h.Cells were incubated with 5 μmol/L DHE for 30 min in the dark.Fluorescence was recorded at Ex/Em wavelengths of 518/ 605 nm respectively using a Bio-Tek FL600 microplate fluorescence reader (Bio-Tek).
T A B L E 1 Sequences of Rat primers and probes used for real-time PCR analysis.

| Senescence-associated β-galactosidase staining
Senescent cells were identified by a Senescence-associated β-galactosidase staining kit (Cell Signaling Technology) according to the supplier's instruction.After incubation, the β-galactosidase staining solution was removed and wells were rinsed in 70% glycerol and images were evaluated on the EVOS xl cell imaging (Thermo Fisher Scientific) microscope (magnification 10x).

| Total RNA extraction and RNA-sequencing
Total RNA was isolated using the RNeasy Mini Kit (Qiagen; 74104) according to the manufacturer's protocol.EVs isolation was performed as described and samples were used for RNA isolation.
RNA concentrations were quantified using the NanoDrop at a wavelength of 260 nm.RNA-sequencing (RNA-seq) was performed on an Illumina HiSeq.2000 instrument by the Genome Sequencing Facility at Novo Gene.
Messenger RNA was purified from total RNA using poly-T oligoattached magnetic beads.After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for nondirectional library.The library was checked inhouse (Novo Gene) with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.Quantified libraries were pooled and sequenced on Illumina platforms, according to effective library concentration and data amount required according to company's protocols (Novo Gene).

| Clustering and sequencing
The clustering of the index-coded samples was performed according to the manufacturer's instructions (Novo Gene).After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generated.

| Statistical analyses
Results are presented as mean ± SEM.All experimental conditions were repeated at least 3 times using cells from different isolations.
Differences were analyzed with Student's t-test for the comparison of two groups and by one-way analysis of variance for the comparison of multiple groups.Statistical analysis was performed with GraphPad Prism 5 (GraphPad Software).A value of p < .05 was considered as significant.

| Characterization of extracellular vesicles derived from hepatocytes
EVs were collected from conditioned medium (serum-free culture medium) of hepatocytes treated with or without FFAs for 48 h by ultracentrifugation (Figure 1A).Isolated EVs samples were diluted in PBS to a final concentration of 10 7 -10 8 particles/mL.
Nanoparticle tracking analysis (NTA) confirmed that the diameter of EVs was on average 99.7-181.3nm with no significant difference between EVs isolated from FFA-or NT-treated hepatocytes (Figure 1B).Western blot analysis showed that CD63 and CD81, the characteristic markers of EVs (NT-EVs treatment and FFA-EVs treatment), were enriched in EVs from hepatocytes (Figure 1C).NTA video samples of FFA-EVs and NT-EVs preparations are depicted in the Supporting Information.
T A B L E 2 Antibodies used in the study.

| Free fatty acid-derived EVs promotes an inflammatory response and induction of senescence in hepatic stellate cells
To further explore the effects of hepatocyte-derived EVs on qHSCs, we examined inflammatory and senescence markers in HSCs.FFA-EVs, but not NT-EVs, induced mRNA expression of IL-1β and IL-6 (Figure 3A).Furthermore, FFA-EVs, but not NT-EVs, increased ROS production in qHSCs (Figure 3B).Since increased expression of IL-6 and IL-1β is also a hallmark of the senescence-associated secretory phenotype (SASP) 22 we hypothesized that FFA-EVs released from hepatocytes decrease HSC activation via induction of senescence.
Indeed, FFA-EVs induced the mRNA expression of the senescence marker p21 but not of p53 (Figure 3C).p21 is used here as an accepted marker of senescence, but it should be noted that p21 and p53 are also involved in other processes such as oncogenic transformation. 23Moreover, FFA-EVs increased the number of senescence-associated (SA)-β-galactosidase positive cells, indicating an increased number of senescent HSCs (Figure 3D,E).

| Extracellular vesicles from free fatty acidtreated hepatocytes (FFA-EVs) promote senescence by targeting AKT/mTOR signaling pathway
The AKT/mTOR pathway is a key regulator of cellular senescence [24][25][26] and it has been shown that activation of the AKT/mTOR/p53 signaling pathway can induce cellular senescence. 27We investigated the effect of hepatocyte-derived EVs on the AKT/mTOR pathway.FFA-EVs, but not NT-EVs, significantly increased the phosphorylation of AKT and mTOR in qHSCs (Figure 4A,B).LY294002, which is an inhibitor of AKT (Figure 4C,D), blocked phosphorylation of both AKT and mTOR with or without EVs treatment.These results suggest that AKT phosphorylation is induced by FFA-EVs in qHSCs and suggest that AKT activity is necessary for mTOR phosphorylation.Furthermore, we assessed the impact of LY294002 on P21 expression and SA-β-galactosidase staining.
We conclude that AKT signaling is necessary for the induction of senescence by FFA-EVs, since LY294002 effectively inhibited both SA-β-galactosidase activity and P21 expression induced by FFA-EVs (Figure 4E,F).The results indicate that AKT/mTOR signaling plays a key role in FFA-EVs-induced senescence, although it cannot be excluded that FFA-EVs also activate other signaling pathways that contribute to senescence.

| RNA sequencing of hepatocyte-derived extracellular vesicles
mRNA sequencing was conducted to determine the cargo of the EVs, in particular to determine differentially enriched mRNAs in FFA-EVs and NT-EVs (Figure 5).380 genes were identified to be increased in the FFA-EV group compared to the NT-EV group (Figure 5A,B).Gene Ontology (GO) enrichment analysis was performed to highlight genome-wide expression patterns. 28GO data of the differentially expressed mRNAs were categorized into 30 functional annotations (p < .05denotes statistical significance between FFA-EVs and NT-EVs).Results of the analysis revealed that these differentially expressed mRNAs are mainly related to phosphorylation, hexose metabolism, and intracellular signal transduction (Figure 5C).Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis 29 indicated that these mRNAs were mainly involved in the following pathways: Herpes simplex virus 1 infection, Rap1 signaling pathway, phospholipase D signaling pathway (Figure 5D), and pathways reported in in vitro MASLD models such as insulin resistance and Notch signaling.The 16 most enriched exosomal RNAs in FFA-EVs compared to NT-EVs are listed in Table 3.

| Differential miRNAs expression profile in extracellular vesicles
Many studies have shown that EV-derived miRNAs regulate target cell functions. 30,31Therefore, we profiled the miRNAs in FFA-EVs and NT-EVs using high-throughput sequencing (miRNA-seq).We identified 12 upregulated and six downregulated miRNAs in FFA-EVs compared to NT-EVs (Figure 6A,B).Furthermore, 362 miRNAs were expressed at equal levels in both types of hepatocyte-derived EVs (Figure 6B).The hierarchical clustering analysis of FFA-EVs and NT-EVs revealed the Metabolic dysfunction associated steatotic liver disease (MASLD) is accompanied by exposure of hepatocytes to excessive levels of (toxic) lipids resulting in hepatocyte damage and death.It is believed that products originating from damaged and/or steatotic hepatocytes contribute to the activation of HSCs during the progression of MASLD [43].
However, the exact mechanism of communication from (damaged) hepatocytes to HSCs and the nature of the compounds released from these damaged hepatocytes is not known.In this study, we used FFA-treated hepatocytes as a model of steatosis and demonstrated that EVs derived from free fatty acid (FFA)-treated hepatocytes attenuate HSCs activation.Furthermore, we observed that the reduced activation correlates with the induction of senescence in qHSCs, but not in aHSCs, via the AKT-mTOR pathway.
Cellular senescence is a state of (near) permanent cell cycle arrest and senescent cells have been identified in MASLD, including senescent 4][35] Increased activity of senescence-associated β-galactosidase (SA-β-gal) can distinguish senescent cells from quiescent cells.Senescent cells also display the senescenceassociated secretory phenotype (SASP), characterized by increased expression of inflammatory cytokines like IL-1β and IL-6 (24).MASLD is associated with inflammatory signaling and increased ROS generation, known to activate NF-κB and both IL-1β and IL-6 are NF-κB-inducible genes.Finally, senescent cells display increased expression of genes like p21 and often, but not always, p53. 34In our study, we observed increased levels of all these senescence markers present in HSCs in early stages of activation.This latter explanation is more likely, since senescence can be induced in fully activated HSCs by, for example, esculetin and doxasozin. 14,36 also demonstrated in this study that FFA-EVs induce the phosphorylation of AKT and its downstream effector mTOR.8][39][40] Moreover, increased AKT signaling and ROS generation have been shown to induce senescence in hepatic stellate cells.Our results support a pro-senescence role of the AKT pathway via its downstream effector mTOR.Our results are therefore in accordance with a pro-senescent and anti-activation role of the AKT-mTOR pathway.It should be noted that NT-EVs did not increase phosphorylation of AKT and mTOR, as well as FFA-EVsinduced markers of senescence, which is consistent with the inhibition of fibrogenesis by induction of senescence.Previously, we have shown that AKT inhibition reverses esculetin-induced senescence of activated HSCs. 14LY294002, an inhibitor of AKT, reversed senescence induced by FFA-EVs.Furthermore, as a positive control for senescence, 41 we utilized rapamycin to test its effects in the presence of FFA-EVs.Rapamycin has been shown to decrease cellular hypertrophy and beta-galactosidase staining. 42Another study showed that rapamycin can extend lifespan in C57BL/6J mice and reverse senescence-related markers. 43These studies demonstrate that rapamycin can directly reverse certain aspects of senescence,  explaining why it has the same effect as LY294002 (see Supporting Information S1: Figure 2).Furthermore, rapamycin can reverse the increase of p-mTOR levels in FFA-EV-treated cells but not in NT-EVtreated cells (data not shown).
This suggests that activation of the AKT-mTOR pathway is necessary to induce senescence of HSCs, although it cannot be excluded that FFA-EVs also activate other signaling pathways that contribute to senescence.
FFA-EVs also induce the SASP, characterized by increased expression of IL-1β and IL-6.This observation is in accordance with the finding that activation of the mTORC1 pathway promotes the SASP.Therefore, we conclude that FFA-EVs induce senescence in quiescent and early activated HSCs in an AKT-mTORC1-dependent pathway.
In MASLD, steatotic hepatocytes release EVs that can target Kupffer cells, sinusoidal endothelial cells and hepatic stellate cells. 44,45EVs are known to contain biologically active molecules, including proteins and various RNA species.The cargo of EVs can be transferred to target cells. 46Specific miRNAs present in EVs can impact on target cells and contribute to the progression of MASLD, for example, miR-122, the most abundant miRNA in hepatocyte EVs, as well as miR-33 and miR-21.EVs from patients with alcoholic liver disease or ethanol-treated hepatocytes contain miRNA-122 which can sensitize macrophages to endotoxin, suggesting polarization toward a M1 phenotype. 47In another study, EVs from hepatocytes from mice with alcohol-induced hepatitis were shown to contain nine upregulated and four downregulated miRNAs. 47A previous study demonstrated that miR-223 was downregulated in HCC tissues and miR-233 inhibited HCC cell proliferation and promoted apoptosis by targeting NLRP3. 48In our study, we applied RNA-sequencing on the EVs from FFA-treated and nontreated hepatocytes to compare their mRNA and miRNA content.We detected 12 upregulated and four downregulated miRNAs in FFA-EVs compared to NT-EVs (Figure 6B).
GO and KEGG pathway analysis revealed that the target genes of these miRNAs are enriched in cellular metabolic processes, the Wnt signaling pathway, the Hippo signaling pathway, the PI3K-AKT signaling pathway, the mTOR pathway and MAPK signaling pathway, implicating that these signaling pathways are targets of EVs-derived miRNAs in MASLD.Moreover, the Wnt/β-catenin, Notch, Hedgehog and Yap/Taz/Hippo signaling pathways are known to be (re)activated in the context of obesity, suggesting important roles in the progression of MASLD to MASH and fibrosis. 52 summary, EVs from steatotic hepatocytes attenuate HSC activation in quiescent/early stage activated HSCs via induction of senescence.This mechanism may attenuate the progression of liver fibrosis in the early stages of MASLD.Activated HSCs appear to be resistant to the effects of FFA-EVs, which may explain why fibrogenesis in advanced stages of MASLD is hard to treat.

F I G U R E 1
Characterization of extracellular vesicles (EVs) derived from hepatocytes.Primary hepatocytes were incubated for 48 h with or without oleic acid and palmitic acid (2:1 ratio) (FFA) to collect medium for the isolation of EVs.(A) Primary rat hepatocytes were treated with or without FFAs for 48 h and EVs were isolated by ultracentrifugation of the conditioned medium.(B) Representative image of concentration and size distribution (nm) of EVs isolated from nontreated hepatocytes, determined by nanoparticle tracking analysis (NTA).(C) Representative Western blot analysis showing the expression of the EVs markers CD63 and CD81 on EVs.Data are shown as mean ± SEM (n = 3).

3. 2 |
Extracellular vesicles from free fatty acidtreated hepatocytes inhibit activation of quiescent hepatic stellate cellsTo investigate whether hepatocyte-derived EVs modulate HSC activation, we tested the effect of EVs from FFA-treated (FFA-EVs) and nontreated (NT-EVs) hepatocytes on both quiescent and activated HSCs.Cells were treated with 15 μg of hepatocyte-derived EVs.Quiescent and activated HSCs were treated with EVs for 72 h, as described in the Materials and Methods.FFA-EVs decreased mRNA expression of the activation markers Acta2 (α-SMA) and Col1α1 in quiescent HSCs (Figure2A,B).NT-EVs slightly decreased Acta2 expression compared to nontreated qHSCs, but this reduction was significantly less than with FFA-EVs.NT-EVs had no effect on Col1α1 mRNA expression in quiescent HSCs.Western blot analysis demonstrated that only FFA-EVs significantly decreased protein expression of α-SMA (Figure2C,D).Both NT-EVs as well as FFA-EVs had no significant effects on activation markers in activated HSCs (Supporting Information S1: Figure1A-D).

F I G U R E 2
Extracellular vesicles (EVs) from free fatty acid (FFA)-treated hepatocytes inhibit activation of quiescent hepatic stellate cells.EVs from steatotic hepatocytes and nontreated hepatocytes were quantified as described in Section 2. (A-D) Quiescent hepatic stellate cells (qHSCs) were treated with 15 μg/well NT-EVs or FFA-EVs for 72 h. mRNA and protein levels of Acta2 were reduced by EVs treatment, and Col1a1 protein level was reduced by treatment with FFA-EVs, but not by NT-EVs.Data are expressed as means ± SEM (n = 3).*p < .05,and ***p < .001compared to control situation.F I G U R E 3 Free fatty acid (FFA)-extracellular vesicles (EVs) inhibit quiescent hepatic stellate cell activation via induction of senescence.Hepatic stellate cells (HSCs) were treated with EVs (15 μg/well) from steatotic hepatocytes and nontreated hepatocytes.Interleukin-1β (IL-1β), IL-6, p21, and p53 mRNA expression were analyzed by quantitative real-time polymerase chain reaction (qPCR), reactive oxygen species (ROS) production was quantified using a DHE-based fluorescence assay.(A) FFA-EVs, but not NT-EVs, increased expression of the SASP/inflammatory markers IL-1β and IL-6.(B) FFA-EVs, but not NT-EVs increased ROS generation in quiescent HSCs (qHSCs).(C) FFA-EVs, but not NT-EVs, increased the mRNA level of the senescence marker p21, but not of p53.(D) FFA-EVs increased the number of SA-β-galactosidase positive qHSCs (magnification 40 x).(E) SA-β-galactosidase positive cells were counted using ImageJ image analysis software and shown as the total number of positive cells per microscopic field.Data are expressed as means ± SEM (n = 3).*p < .05,and **p < .01compared to control situation.differentially expressed miRNAs.Based on the heat map which showed miRNA expression (Figure 6B), both downregulated miRNAs (miR-223-p, miR-139-5p, miR-28-3p, miR-351-5p, miR-125a-3p, miR-322-3p), as well as upregulated miRNAs (miR-205, miR-292-5p, miR-96-5p, miR-194-5p, miR-16-5p and miR-451-5p) were observed.GO terms revealed that differentially expressed mRNAs could be categorized into 20 functional annotations (biological process (BP), cellular component (CC) and molecular function (MF) (Figure 6D).KEGG pathway analysis indicated that the PI3K-AKT signaling pathway and mTOR signaling pathway are involved (Figure 6E), supporting our Western blot results depicted in Figure 4.4 | DISCUSSION Liver fibrosis is the result of an excessive wound-healing response in chronic liver injury and is characterized by the excessive deposition of extracellular matrix (ECM) components such as collagens in the F I G U R E 4 Free fatty acid (FFA)-extracellular vesicles (EVs) promote senescence by targeting the AKT/mTOR signaling pathway.Quiescent hepatic stellate cells (qHSCs) were treated with EVs (15 μg/well) from steatotic hepatocytes and nontreated hepatocytes.(A, B) qHSCs were treated with EVs (NT and FFA groups) and harvested after 72 h.FFA-EVs significantly increased the levels of p-mTOR and p-AKT.(C, D) qHSCs were treated with EVs (NT and FFA groups) with or without LY294002 and harvested after 72 h.LY294002 significantly reduced the phosphorylation of both AKT and mTOR.(E) LY294002 reduced the number of SA-β-galactosidase positive cells in FFA-EV-treated qHSCs (magnification 10×).(F) SA-β-galactosidase positive cells were counted using ImageJ image analysis software and shown as the total number of positive cells per microscopic field.(G) LY294002 significantly decreased P21 expression in FFA-EV-treated qHSCs.Data are expressed as means ± SEM (n = 3).*p < .05,**p < .01 and ***p < .001compared to control situation.liver, disrupting normal liver architecture and function.Activated hepatic stellate cells (aHSCs) are the main producers of these ECM components.In chronic liver injury, quiescent HSCs transdifferentiate into aHSCs, acquiring a myofibroblast-like phenotype.32

F I G U R E 5
Extracellular vesicles (EVs) mRNA expression profile of free fatty acid (FFA)-EVs and NT-EVs.(A) Volcano plots of enriched mRNAs in FFA-EVs compared to NT-EVs.(B) Heat-map of mRNA expression levels for selected genes at baseline.(C) Gene Ontology (GO) analysis.(D) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.NT-EVs group (n = 5), FFA-EVs group (n = 3).inFFA-EV-treated qHSCs, but not in FFA-EV-treated aHSCs.It should be noted that the treatment time of qHSCs was 72 h implying that at the end of the treatment the qHSC are intermediately activated.The fact that steatotic hepatocytes release EVs that attenuate HSC activation in MASLD may appear to be counterintuitive at first, however, it should be noted that many more cells are involved in the inflammatory and fibrotic process in advanced stages of MASLD, for example, Kupffer cells, infiltrating inflammatory cells and sinusoidal endothelial cells that all impact on the microenvironment in the liver.Furthermore, FFA-EVs only impact on early stage activated HSCs.Completely activated HSCs are resistant to the effects of FFA-EVs.Therefore, the release of FFA-EVs that attenuate fibrosis in early stage steatosis may act as a protective mechanism.Finally, senescent cells, including HSCs, display an inflammatory phenotype (SASP) which may also, indirectly, promote fibrosis.More studies are necessary to elucidate the exact role of different cell types on the fibrotic process in the advanced stages of MASLD.At present, we do not have an explanation for why aHSCs are less susceptible to the senescence-inducing effect of FFA-EVs.It is possible that in fully activated HSCs there is a point of no return that prohibits reversal or attenuation of activation.Indeed, fully activated HSCs produce autocrine growth factors, leading to uncontrolled and irreversible HSC proliferation.Alternatively, the FFA-EVs contain factors that interfere with targets that are only

F I G U R E 6
Differential miRNAs expression profile in extracellular vesicles.(A) Venn diagram showing the unique and overlapping miRNAs presented in NT-extracellular vesicles (EVs) and free fatty acid (FFA)-EVs.(B) Heat-map showing scores of miRNAs from NT-EVs and FFA-EVs.Red represents upregulated genes and blue represents downregulated genes.(C) Volcano plots showing numbers of up or downregulated genes.(D) Gene Ontology (GO) analysis.(E) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.NT-EVs group (n = 5), FFA-EVs group (n = 3).
T A B L E 3 Top 16 enriched mRNAs in FFA-EVs released from hepatocytes (p < .05).