Animal model and treatments
Common bile duct ligation rat model (CBDL). The common bile duct ligation operation of rat is one of the typical HPS model with a well-established methodology[3, 30]. All animal experiments were approved by the Animal Care Committee of Third Military Medical University, Chongqing, China. Male Sprague-Dawley rats (200-220g, 6-7 weeks age, 30 rats each group) were anesthetized with intramuscular injections of ketamine (80mg/kg) and xylazine (10mg/kg). The control rat was sham-operated by isolation of the common bile duct without ligation. The lung of the animals was dissected and analyzed after 1 week, 3 week and 5 weeks after surgery. Blood sample was aseptically drawn from the abdominal aorta during laparotomy. A 0.2ml sample of arterial blood was collected into a heparinized gas capillary tube to measure the arterial gas levels. Serum was separated from the blood samples (centrifugation at 2000×g, 4°C), then was used to separate exosomes.
Arterial blood gas analysis and sample preparation. Arterial blood was collected from the abdominal aorta under chloral hydrate anesthesia (40 mg/kg IP, Nembutal, Ceva Sante Animale), and blood gas analysis was performed in the Laboratory of Clinical Biology (Southwest Hospital, China). Hypoxemia was defined as a PaO2 < 80 mmHg. Additionally, serum was prepared by centrifugation of the blood at 10000 rpm for 10 min at 4°C and stored at −80°C prior to analysis.
Exosome treatment. To analysis the function of HPS exosome toward rat lung, exosomes isolated from sham rat serum, HPS rat serum, MIBECs cell line and MIBECs with PICALM-AU1 overexpression (method as described below) were injected into healthy rats (200-220g, 6-7 weeks age). Rats were randomly divided into four groups (ss-Exo, sham-serum exosome; Hs-Exo, HPS serum exosome; ct-Exo, MIBECs-derived Exo; PO-Exo, PICALM-AU1 OE MIBECs-derived exosome). Exosomes (100µg total protein in 100µL volume) were injected three times and once every other day via the caudal vein. Exosomes from sham and HPS rat serum were used to treat PMVECs to analysis EndMT in cell line.
Virus treatment. To analysis the function of PICALM-AU1 toward rat lung, lentivirus with PICALM-AU1 overexpression and knockdown was constructed. The LV-NC, LV-PICALM-OE and LV-PICALM-KD viruses were injected into healthy rats (200-220g, 6-7 weeks age) via caudal vein (each at 100µL of 2×1010TU/ml). After two weeks, the rats were carried on CBDL operation; To investigate the function of exosomal-PICALM-AU1 toward rat lung, lentivirus LV-NC and LV-PICALM-OE were treated MIBECs (each at 10µL of 1×109TU/mL). After 72hr, detection of the PICALM-AU1 expression. And then, exosome was isolated for rat infection; To analysis the function of PICALM-AU1 toward PMVECs, LV-NC, LV-PICALM-OE and LV-PICALM-KD viruses were treated PMVECs (each at 10µL of 1×109TU/mL). After 72hr, detection of the gene expression, protein synthesis was carried on.
Total RNA of CBDL operation group and sham group rat liver was extracted and transcribed. Double-stranded cDNA was labeled using the Quick Amp Labeling Kit (Agilent Technologies Inc, USA) and hybridized to the Array star Rat 8×60K lncRNA Array, version 2.0. Following the washing steps, the arrays were scanned with the Agilent Scanner G2505B, and the array images were analyzed using Agilent Feature Extraction software, version 10.7.3.1. Quantile normalization and subsequent data processing were performed using GeneSpring GX software, version 11.5.1 (Agilent Technologies Inc, USA). Volcano plot filtering was used to identify the lncRNAs with statistically significant differences, and the threshold to screen upregulated or downregulated lncRNAs was identified at a fold change of 1.5 or greater and a P value of 0.05 or less.
Tissue harvest and Histology
Liver and lung samples were fixed in 4% phosphate buffered formaldehyde solution (Klinipath, Belgium), dehydrated, embedded in paraffin and stained with Hematoxylin and eosin, Masson staining, Immunohistochemistry, Immunofluorescence and Fluorescence in situ hybridization. All of the antibody information was as followed in Table S2.
H.E staining of rat lung tissue was according to previous study . Immunohistochemical staining on lung tissue allowed to quantify protein expression levels. Specific anti-VWF, anti-VE-cadherin and anti-Vimentin were used. Slices that underwent immunostaining with omission of primary antibodies or with IgG were used as negative controls. Paraffin-embedded lung sections (5 µm thickness) were deparaffinized, rehydrated by serial immersion in ethanol, and pretreated with citrate buffer. Non-specific binding sites were blocked via incubation in 3% H2O2 (Merck, Germany) and BSA respectively. Epitope detection was performed using the ultraView Universal DAB Detection Kit (Dako, Denmark). Counterstaining was performed with hematoxylin.
The vascular density of specimens stained for VWF was measured semi-quantitatively using Cell Software (Olympus, Japan). Results are expressed as mean positively stained area (% ± SE) per field. The number of vascular per high power field (objective 40×) was counted in 15 randomly selected fields for each rat, and the mean value of the vascular in these fields was calculated (mean number of vascular per field ± SE). All final histological scores are represented as the mean of the scores determined by two independent researchers, who were blinded to the study samples.
Immunofluorescence. For immunofluorescent double staining, paraffin-embedded lung sections (5 µm thickness) or cell slides were deparaffinized, rehydrated by serial immersion in ethanol and pretreated with EDTA, followed by incubation in 50 mM NH4Cl, 0.1% Triton X-100 and 1% BSA. Anti-VE-cadherin, anti-Vimentin, anti-ZEB1 and anti-ZO1 were used as primary antibodies. Slices that underwent immunostaining with omission of primary antibodies or with IgG were used as negative controls. The binding sites of the primary antibodies were revealed with Alexa Fluor-594 goat anti-rabbit and Alexa Fluor-488 goat anti-mouse secondary antibodies (Invitrogen, USA). Nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI) (Life Technologies, USA). Samples were visualized with a fluorescence microscope (Olympus, Japan).
FISH (Fluorescence in situ hybridization) combined with fluorescent IHC staining
FISH targeting PICALM-AU1 in rat liver tissue sections was performed using a commercially available RNA scope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, USA) by following the manufacturer’s instruction. Fluorescent IHC staining target PICALM-AU1 was performed after FISH staining as described in the above section (Histopathology, Masson’s Trichrome staining, and immunohistochemistry). Zeiss LSM 700 confocal laser scanning microscopy were used to visualize FISH results (Carl Zeiss, Germany).
cDNA synthesis and qPCR
LncRNA, miRNA and mRNA expression were analyzed in total RNA from tissue and cell samples using the Applied Biosystems 7000 sequence detection system (Applied Biosystems, UK) with SYBR Green and the comparative CT method. Values were reported relative to the endogenous control glyceraldehyde-3-phosphate dehydrogenase. All amplification reactions were performed in three independent times. Primer sequences were described in Supplementary Table S1.
Protein expression was determined by Western blot in rat lung and PMVECs samples as previously described . Antibodies information was shown in Table S2. Blots were visualized by ECL reagents (DAKO, Denmark), and digital images were taken using a luminescent image analyzer LAS-4000 (General Electric, UK). β-actin was used for the normalization of quantitative densitometry values.
Cell culture and in vitro experiments
Rat pulmonary microvascular endothelial cells (PMVECs) and mice intrahepatic biliary epithelial cells (MIBECs) were purchased from American Type Culture Collection (ATCC Cell Biology Collection, USA). And cells were maintained at 37°C in RPMI medium (Gibco, USA) supplemented with 10% fetal bovine serum (Invitrogen, USA). For cell transfection experiments, cells were seeded at 60-70% confluence. Vectors were mixed with Lipofectamine 3000 (Promega, USA), diluted in EGM2, and treated for 24h, as described . After 24h, cells were treated for miR144-3p mimics/ inhibitor or sham/HPS exosome. Then, Luc activity was detected by Dual-Luciferase Reporter Assay System (Promega, USA) in the GloMax-Multi Detection System Photometer (Promega, USA). Assays was carried out three times independently and the average Luc activity levels were presented as mean ± SE.
HPS patient specimens
We selected the HPS patients and collected the blood. Then, exosome was isolated for detecting gene transcript. This study was conducted according to the ethical guidelines of the Declaration of Helsinki. It was approved by the U.S. National Library of Medicine, Clinical Trails.gov (https://clinicaltrials.gov/, NCT:03435406). All participants provided written informed consent and agreed to the publication of their anonymous information. Patients were screened for the presence of HPS. HPS was diagnosed by three parameters: (1) presence of cirrhosis (2) positive contrast-enhanced echocardiography, and (3) an alveolar-arterial oxygen gradient (P(A-a) O2) ≥ 15 mmHg (or ≥20 mmHg in patients >64 years). Intrapulmonary vascular dilations were assessed by contrast-enhanced echocardiography. Agitated saline causes microbubbles of >10 µm in diameter that usually do not pass through the pulmonary capillary bed. Appearance of microbubbles, after injecting in a peripheral vein, first in the right heart, and within three to six heart actions in the left heart demonstrates abnormal vasodilation of the intrapulmonary capillary bed. Early (<3 heart beats) appearance of microbubbles in the left heart was considered as intracardiac shunting. These patients were excluded from this study as the presence or absence of intrapulmonary shunting could not be judged by contrast-enhanced echocardiography.
Exosome Isolation and characterization
Human patient serum, rat serum and MIBECs medium were collected by centrifugation at 2,000 g for 15 min followed by 16,000 g for 20 min at 4°C. Then, the supernatants were collected and ultracentrifuged at 110,000 g for 70 min. Afterwards, pellets were resuspended in sterile PBS and purified by centrifugation at 110,000 g for 1h. Subsequently, the exosomes were resuspended in PBS and filtered through 0.22 µm filter (Millipore, USA), stored at -80°C for further analysis.
To characterize the morphology of the isolated exosomes, transmission electron microscopy (Hitachi HT7700, Japan) was used. For size distribution of the isolated exosomes, qNano (Izon Science, New Zealand) was used following the manufacturer’s instructions. To analyze the protein markers of exosomes, Western blotting assays were performed, and anti-CD63, anti-CD86 antibodies were used.
Results were obtained from at least three independent experiments and are expressed as mean±SD. Data were analyzed by two-tailed student t test, one way-variance analysis with Tukey’s post-hoc test or linear regression using GraphPad Prism software version 8.0 (GraphPad Software Inc., USA). A P value of ≤ 0.05 was considered statistically significant.