Reagents
The chemicals used in our experiments were: Aldosterone (Sigma-Aldrich, A9477), Spironolactone (Sigma-Aldrich, S1200000), bafilomycin A1 (Selleck, S1413), GW4869(Selleck, S7609), Collagenase, Type IV (sigma, 17104019), VEGF(Peprotech, AF-450-32), CD63-tdTomato Lentifect (GeneCopoeia, LP772), Glutaraldehyde 25% (Nacalai, 17003-92), Aldosterone ELISA KIT(Elabscience, E-EL-0070c).
Patients
Fibrotic liver specimens were obtained from 10 patients with liver fibrosis due to bile duct stones. Normal liver specimens were obtained from 10 patients due to partial liver resection from hepatic hemangioma resection. The serum was obtained from 23 patients with liver fibrosis, and 6 from healthy volunteers. All patients informed written consent and the Ethics Committee at the local hospital approved the use of samples.
Animal experimental design
Sprague-Dawley (SD) rats were provided by Guangdong Medical Laboratory Animal Center (GDMLAC) and approved by the Southern Medical University Experimental Animal Ethics Committee(K2017038). Rats were housed under 12 h light/12 h dark and 22-24oC, with water provided. 63 male SD rats were randomly divided into 7 groups respectively (control, Aldo, Aldo+Spi, Aldo+AAV si-NC, Aldo+AAV si-ATG5, si-NC, si-rab27a, n=9). The Aldo groups were treated with Aldo(0.6ug/g/d) continuously via osmotic micro-pumps (Alzet, 2004) for 28 days. Spi was given to rats orally(10mg/kg/d). Adeno-associated virus (AAV) was administered by intravenous injection.
Cell lines and culture conditions
Culture media and supplements for cell culture were from Gibco. Immortalized human-liver endothelial cell lines (TMNK-1) were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank). LX-2 was from iCell Bioscience Inc, Shanghai. Cell lines were cultured in DMEM with 10% FBS.
LSECs and HSCs isolation, identification, and treatment
Primary rat LSECs were isolated from SD rats, based on the modified method[13]. Anesthetizing rats with heparin and then inserted Y integrated catheter into the portal vein and secured, perfusing with 150 ML HBSS ad 5% FBS without calcium at a flow rate of 15ml/min and immediately cutting the inferior vena cava. Next, add 100 ML collagenase solution at a flow of 5ml/min. Removing the liver and digesting it in collagenase solution. After removing the parenchymal cells, use 20% Histodenz to separate LSECs and HSCs. The LSECs were plated on collagen-coated plates with a medium comprising 40% MCDB131, 40% 1640, and 20% FBS with VEGF supplement, while the HSCs were cultured with 1640 with 20% FBS supplement. The LSECs were identified by an SEM test and expression of Stab2 protein, and the HSCs were identified by expression of Desmin protein.
Extracellular vesicles isolation, quantification, and identification
The cell culture medium will be removed serum or any other components that may contain EVs before collection. After the administration of stimulation, cell culture supernatant was collected on ice and pre-cleared from debris by sequential centrifugation (300g for 10min, 2000g for 10min, and 10,000g for 30min). The prepared fluid was further by ultracentrifugation at 100,000g for 2h. The pellet was resuspended in PBS and ultracentrifuged again at 100,000g for 2h. The extracellular vesicles were quantified by the BCA protein determination method and NTA (using a Particle Metrix’ ZetaView according to the manufacturer’s instructions, the EVs sample were diluted in serum-free PBS and injected into the beam path to capture movies of EVS), and identified by TEM and expression of TSG101, Alix, CD9, and CD63.
Western blotting
The protein expression was detected by western blot. The primary antibodies included Anti-STAB2(ABclonal, A10074), Anti-CD34(Bioss, bs-0656R), Anti-LAMP2(proteinech, 66301), Anti-CD63(proteintech, 25682), Anti-TGF-Beta 1 (proteintech, 21898), Anti- α-SMA(Boster,BM0002 ), Anti-TSG101 (proteintech, 14497), Anti-CTGF(Abcam, ab6992), Anti-SQSTM1(abcam, ab56416), Anti-LC3B(CST, 2775S). The secondary antibodies were donkey anti-mouse (1:15000, LI-COR biosciences, C40910-04) and goat anti-rabbit (1:15000, LI-COR biosciences, C510070-08).
q-RT-PCR
The total RNA of cells (2*106) or liver tissue was extracted by EZBioscience (B0004D). cDNA was produced with HiScript III RT SuperMix for qPCR (Vazyme, R323-01) and 50ng of cDNA was used for q-RT-PCR with Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Q712-03). The fold change related to β-actin was calculated using the 2-DeltaDeltaTh method.
Histological analysis
Paraffin sections (4 μm) of human and mouse liver tissues were processed with hematoxylin and eosin (H&E) (DH0040/DH0054; Leagene Biotechnology) and Masson staining (MST-8004; Fuzhou Maixin Biotechnology). Paraffin-embedded sections were incubated overnight with primary antibodies and incubated with horseradish peroxidase-conjugated secondary antibodies. Immunohistochemical staining was observed using 3,3′-diaminobenzidine (No. ZLI-9017; Zhongshan Biotech, Beijing, China).
Measurement of intralysosomal function
Lysosomal pH was measured with LysoSensor Yellow/Blue DND-160(Yeasen, 40768ES50) based on Zoncu, R., Bar-Peled, L., Efeyan, A., Wang, S., Sancak, Y., and Sabatini, D.M. (2011). mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H (+)-ATPase. Science 334, 678–683.
Wound healing assay
Confluent cultures of cells were placed in a serum-free medium. Wounds were then struck through the cells using a pipette tip and the medium on the cells was replaced a third time. 24h later, the cell was imaged using phase contrast microscopy. The prolonged time of wound closure for each condition was determined using ImageJ software.
Migration Assay
Transwell migration was performed by using cell culture inserts (corning, 3470). After removing the cells on the top of the Transwell insert, cells were stained with crystal violet and counted from microphotographs of five different fields.
Gel contraction
Prepared the collagen lattice with the type I rat tail collagen. HSCs were then implanted on the collagen lattice. After 48h of culture, the lattice area of collagen was measured and compared in each group.
5-Ethynyl-20-deoxyuridine (EdU) Assay
Cell proliferation was detected by the EdU kit (Cell Light EdU DNA imaging kit, RiboBio, Guangzhou, China) according to the manufactory's instrument. Images were detected by a fluorescent microscope. The average ratio of EdU-stained cells (green) to Hoechst-stained cells (blue) was used to evaluate cell proliferation activity.
Cell counting kit-8 (CCK8)
Cell proliferation assay was also performed using the Cell Counting Kit-8 (CCk-8, Dojindo, Kumamoto, Japan). Cells were plated in 96-well plates at 1*104 cells per well. CCK-8 was added in each well at 24, 48, and 72h, and after an additional 2h culture, the absorbance of each well was detected and calculated.
Transmission Electron Microscopy (TEM)
Cells were prepared and fixed with 2.5% glutaraldehyde solution for 24h at 4°C, then treated with 2% uranyl acetate solution for 2 h. After dehydration in 50%, 70%, 90%, and 100% acetone, cells were embedded to make ultrathin sections for observation under an electron microscope (Hitachi H-7500). For negative staining of EVs, pipette the EVs sample on the copper mesh, and absorb the excess fluid with paper after 3min. Add phosphotungstic acid (3%, pH 7.0) to the copper mesh dropwise, and then use paper to absorb the excess dye solution from the edge of the copper mesh after 3min. Add 3 drops of pure water to the copper mesh, and use filter paper to draw excess water from the edge of the copper mesh. After drying, the negative staining sample was imaged by transmission electron microscopy (Hitachi H-7650).
Scanning Electron Microscopy (SEM)
Cells were fixed with 2.5% glutaraldehyde and subsequently dehydrated and then coated with gold using the coating apparatus. The fenestrae of cells were observed with SEM at 3.0-kV acceleration voltage.
Mass spectrometry
Cells were collected and lysed in lysis buffer, and the protein concentrations of the samples were determined by Bicinchoninic Acid assay, and an equivalent amount of each sample was processed through electrophoresis for protein quantities, enzymatic hydrolysis, labeling, and mixing, High pH reversed-phase chromatography (peptide fractionation), liquid phase tandem mass spectrometry analysis, and finally get mass spectrometry raw data. Then, the raw data were further processed with MaxQuant software.
Extracellular vesicles RNA-sequencing
Total RNA was extracted from EVs. For small RNA, 3’ and 5’ adaptors were added to each end, followed by reverse transcription and PCR amplification. cDNA fragments from 18 to 30 nucleotide RNA were purified and then sequenced on Illumina HiSeq 2500 sequencer. The data is routinely processed and validated. For lncRNA and mRNA, cDNA was purified and then sequenced on Illumina PE150 sequencer.