Isolation and characterization of exosomes
This study was approved by the Institutional Review Board for Human Research at the Medical University of South Carolina. Human EPCs were isolated from the cord blood from a healthy pregnant woman and were cultured as previously described . Briefly, the cord blood samples were collected from the umbilical veins during normal full-term vaginal deliveries, and informed consent was obtained from the mother for all cord blood collections. EPCs were cultured in endothelial basal medium (EBM-2; Lonza, Allendale, NJ, USA) supplemented with EBM-2 SingleQuots (Lonza, Allendale, NJ, USA) containing 10% exosome-depleted fetal bovine serum (FBS; System Biosciences, Palo Alto, CA, USA), 1% penicillin, and streptomycin (GIBCO, Gaithersburg, MD, USA) for 48 h, while NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO, Gaithersburg, MD, USA) containing 10% exosome-depleted FBS (System Biosciences, Palo Alto, CA, USA), 1% penicillin, and streptomycin (GIBCO, Gaithersburg, MD, USA) for 48 h. Medium was harvested and centrifuged at 2000×g for 30 min to remove cells and debris. Exosomes were then isolated from the cell-free medium using the Total Exosomes Isolation Kit following the manufacturer’s instructions (Invitrogen, Asheville, NC, USA) and re-suspended in PBS.
The total protein concentration of the exosomes was measured by detergent-compatible (DC) protein assay (Bio-Rad, Hercules, CA, USA). The size distribution and the total number of exosomes were analyzed by nanoparticle tracking analysis (NTA) with ZetaView PMX 120 (Particle Metrix, Meerbusch, Germany). Exosome markers, such as tetraspanin proteins CD9, CD63, and CD81, were determined by western blot. Each experiment was carried out in triplicate.
Lipopolysaccharide-induced acute lung injury model
Previously, we reported that EPC exosomes exert protective effects in a cecal ligation and puncture model which is a clinically relevant murine model of sepsis. To further explore the effects of EPC exosomes in a murine ALI model, intratracheal instillation of LPS was used to induce ALI. Investigations conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH and were approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina. CD-1 outbred mice (aged 7–8 weeks) were housed in a pathogen-free environment. The mice underwent intratracheal instillation of either 25 μg LPS diluted in 75 μl PBS as described previously  or 75 μl PBS. Four hours after acute lung injury induction, the mice were treated with 70 μg of EPC exosomes or negative control NIH3T3 exosomes or PBS separately through intratracheal administration. Thus, four experimental groups were created: (1) PBS control, (2) LPS+PBS, (3) LPS+EPC-exo, and (4) LPS+3T3-exo. Subsequent experiments examined three to seven mice per group. Bronchoalveolar lavage fluid (BALF) and perfused lung tissues for myeloperoxidase (MPO) activity and Evans blue assay were collected at 24 h as described below, and formalin-fixed paraffin-embedded histological lung tissues were collected at 48 h after lung injury. All the samples were stored at − 80 °C or 4 °C until analysis.
Assessment of lung inflammation
Lung inflammation was compared between experimental groups using the following methods: (1) measurement of BALF cell count, (2) measurement of BALF cytokines and chemokines, and (3) measurement of lung tissue myeloperoxidase activity. BALF was collected from mice in each group 24 h after LPS instillation. After euthanasia, the thorax was opened to expose the trachea. The trachea was cannulated with a 20-g angiocatheter and lavaged four times with cold PBS (0.75 ml) using a 1-ml syringe. The BALF was centrifuged at 600×g for 5 min to pellet cells. The cell pellet was re-suspended in 500 μl red blood cell lysis buffer and centrifuged at 600×g for 5 min. Cell pellets were re-suspended in 500 μl PBS, and immune cells were quantified using a Countess II Automated Cell Counter (Thermo Fisher Scientific, Waltham, MA, USA). The supernatant was collected and analyzed for cytokine and chemokine levels using the pro-inflammatory focused 32-plex (Eve Technologies, Calgary, AB). The remaining supernatant was aliquoted and frozen at − 80 °C for additional experiments.
Myeloperoxidase activity was determined in lung tissue as an index of neutrophil accumulation as previously described [17, 18]. Briefly, lung tissues were perfused, weighed, and homogenized in 1 ml potassium phosphate buffer (50 Mm, pH 6.0). The homogenized tissues were centrifuged for 15 min at 10000 rpm, and the supernatant was discarded. The tissues were re-suspended in 1 ml potassium phosphate solution (50 mM) containing 0.5% hexadecyl-trimethylammonium and sonicated for 20 s. The samples were frozen and thawed twice and centrifuged for 10 min at 10000 rpm. The supernatants (10 μl) were combined with 80 μl 0.75 mM H2O2 (Sigma, St. Louis, MO, USA) and 110 μl TMB solution (2.9 mM TMB in 14.5% DMSO and 150 mM sodium phosphate buffer at pH 5.4), and the plate was incubated at 37 °C for 5 min. The reaction was stopped by adding 50 μl H2SO4 (2 M, Sigma, St. Louis, MO, USA), and the absorption was measured at 450 nm. The quantification of MPO was calculated from a MPO standard curve and was expressed in units per gram of the tissue.
Measurement of lung vascular leak
Lung vascular leak was measured in each experimental group using (1) Evans blue assay in lung tissue, (2) lung water content, and (3) BALF protein concentrations. The Evans blue dye assay was performed as described previously . Briefly, the mice were administered 1% Evans blue dye solution (Sigma, St. Louis, MO, USA) in saline via tail vein injection. After 40 min, the mice were sacrificed and perfused via the heart, and the lung tissues were collected. The lung weights were measured and placed in 1 ml of formamide (Avantor, Center Valley, PA, USA) at 60 °C for 24 h to extract Evans blue dye. The samples were centrifuged at 2000 rpm for 10 min, and the supernatants were collected. The concentrations of Evans blue dye in the supernatants were quantified by measuring absorbance at 620 nm and calculated from a standard curve by a plate reader.
For lung water content, the left lung was harvested and weighed to measure a wet weight in each group. The wet lung was then dried in an oven at 60 °C for 48 h and re-weighed as dry weight. The lung water content was calculated as the ratio of wet weight to dry weight. Protein levels in the BALF supernatant were determined by DC protein assay (Bio-Rad, Hercules, CA, USA).
Lung histology and lung injury score
The lung tissues were collected from mice at 48 h after LPS instillation. The lungs were inflated with 10% buffered formalin, fixed with 10% buffered formalin, embedded in paraffin, and cut into 5-μm sections. Tissue sections were stained with hematoxylin and eosin (H&E), evaluated, and scored by a pathologist who was blinded to the experimental groups. To evaluate the lung jury, seven independent random lung fields were evaluated per mouse for neutrophils in alveolar spaces, neutrophils in the interstitial spaces, hyaline membranes, proteinaceous debris filling the airspaces, and alveolar septal thickening and weighted according to the relevance ascribed by the official American Thoracic Society workshop report on features and measurements of experimental acute lung injury in animals . The resulting injury score is a continuous value between 0 and 1.
Human small airway epithelial cell culture and miR-126 transfection
To determine the potential effects of exosomal miR-126 on epithelial gene expression, small airway epithelial cells (SAECs) were transfected with synthetic miR-126. SAECs were cultured in human collagen type IV-coated flasks (Sigma, St. Louis, MO) supplemented with SAGM Bullet kit culture medium (Lonza, Allendale, NJ, USA). Cells were seeded into 12-well plates and then transfected with a miR-126-3p mimic (40 nM), miR-126-5p mimic (40 nM), or control miRNA (40 nM) for 48 h using human airway epithelial cell avalanche transfection reagent according to the manufacturer’s instructions.
At 24 h after transfection, the cells were then stimulated with LPS (100 ng/ml, Sigma, St. Louis, MO, USA) for 24 h. The total RNA was extracted from cells, which was used to do RNA sequencing analysis as well as measurement of mRNA levels by real-time polymerase chain reaction (RT-PCR) described below.
RNA sequencing and pathway analysis
To determine the effect of miR-126-3p and miR-126-5p on gene expression profile in SAECs, we transfected SAECs with miR-126-3p and miR-126-5p or control siRNAs for 48 h and total RNAs were isolated using RNeasy plus kit (QIAGEN, Germantown, MD, USA) following the manufacturer’s instructions. Extracted RNA was used to prepare next-generation sequencing (NGS) libraries, and the sequencing was performed on an Illumina HiSeq 2500 instrument at the MUSC Genomic Sequencing Core Facility or by Novogene (Chula Vista, CA, USA). The pathway analysis was performed with the Genomic Sequencing Core Facility.
Total RNA extracted from SAECs was also used to perform RT-PCR to validate differentially expressed mRNA identified in the RNAseq analysis. The RNA (10 μl per reaction) was reverse transcribed using the High Capacity cDNA Reverse Transcription Kits (Thermo Fisher Scientific, Waltham, MA, USA). Following cDNA synthesis, the levels of mRNA were determined by CFX96 Real-Time PCR system (Bio-Rad, Hercules, CA, USA) using SYBR green qPCR master mix (QIAGEN, Germantown, MD, USA) according to the manufacturer’s instructions. Data were analyzed with 2−ΔΔCt value calculation, using GAPDH for normalization.
EPC exosomes and NIH3T3 exosomes were lysed with ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer (Abcam, Cambridge, MA, USA) containing protease and phosphatase inhibitors (Cell Signaling, Boston, MA, USA). All lysed samples were kept on ice for 30 min and centrifuged for 10 min at 4 °C at 10,000×g. The cell lysates were collected, and protein concentrations were measured using a DC protein assay (Bio-Rad, Hercules, CA, USA). Approximately 20 μg of exosomes protein was loaded into each lane for western blot. All exosome-specific primary antibodies including anti-CD9, anti-CD63, and anti-CD81 (System Biosciences, Palo Alto, CA, USA) were used at 1:1000 dilution, and exosome-validated peroxidase-labeled secondary antibody was at 1: 20000 dilution. The immunoreactive protein bands were visualized by ECL detection kit (GE Healthcare, Pittsburgh, PA, USA) and analyzed using ImageJ software.
The in vitro experiments were performed at least three independent times. The data were analyzed using GraphPad Prism 7.01 software and represented as mean ± SE. Means of multiple groups were compared by one-way analysis of variance (ANOVA). Independent sample t test was performed to compare the means between two different groups. A value of p < 0.05 was considered statistically significant.