Ethical approval
In the present study, all experiments followed the Guide for Care and Use of Laboratory Animals by America (National Institute of Health) and approved by Army Medical University’s Ethics Committee and Research Council and Animal Care and Use Committee (Number. DHEC-2012-069).
Animals’ preparation and sepsis model
Male and female Sprague–Dawley (SD) rats (with a weight range of 195 ± 10 g) were purchased from the Animal Research Center of the Daping Hospital (Army Medical Center). Then sepsis model was performed by cecal ligation and puncture (CLP) procedure as described [28, 29]. In brief, laparotomy was performed under anesthesia with pentobarbital sodium solution (45 mg/kg intraperitoneal), the cecum of rats was gently exposed, the feces were concentrated at the end of the cecum, then the surgical silk was used to ligate the end of the cecum. Afterward, rectangular needle was used to perforate the bling-end of the cecum, and the exposed intestine was placed back to abdominal cavity, then the abdomen was closed by interrupted suture. Finally, each of the rats was injected with 5 mL of normal saline intraperitoneally, and they were subsequently fed with water until use.
Cell culture and reagents
Pericytes (PCs) were obtained from the retina of weanling SD rats [30]. The eyeballs were removed immediately after general anesthesia, and placed in PBS containing 1% penicillin/streptomycin. Afterward, the eyeball was separated from sclera and pigment epithelium using scissors and tweezers. Next, it was gently placed in the dissection buffer. The retina was snipped and digested with 2 mg/mL type I collagenase, and then DMEM containing 20% FBS was added to stop the digestion. After filtration and centrifugation, the mixed medium was cultured in 5 mL pericyte specific medium (ScienCell, America) in the presence of corresponding pericyte growth supplement (ScienCell, America) and antibiotics (ScienCell, America).
Vascular endothelial cells (VECs) were obtained from the left lung of rats as described [31]. In brief, thoracotomy was performed under anesthesia with pentobarbital sodium solution (45 mg/kg intraperitoneal). Later the pulmonary vein in the left lung was exposed gently and subsequently took out, and the vein was washed for 4 times and cut to pieces, then the vein pieces were placed in the culture flask and cultured (DMEM-F12 medium, 10% serum (Gibco, America).
Several studies have pointed out that, endotoxemia could be used to model the acute inflammatory response associated with sepsis and LPS was the major Gram negative bacteria [32, 33]. As a result, LPS was used as a stimulation in vivo.
LPS (055:B5) was purchased from Sigma (America). CCK8 Detection Kit was purchased from Dojindo (Japan), and TNFα Elisa Kit was purchased from Abcam (America). ERK1/2 agonist and ERK1/2 inhibitor were purchased from Selleck (America). STAT3 agonist was purchased from MedChemExpress (America). Anti-CD44 blocking antibody was purchased from Abcam (America).
Harvest of MVs
MVs were harvested from cultured PCs supernatant [34]. Before the MV harvest, PCs were starved by serum-free basal medium for 24 h, then the supernatant was collected in sterile centrifuge tubes. The supernatant was processed as follows: 500 g for 20 min (4 °C, discard the precipitation), then 2000 g for 25 min (4 °C, discard the precipitation), then 20,000 g for 80 min (4 °C, discard the supernatant). The MV pellet was washed by PBS and stored at − 80 °C until subsequent analysis [35].
Flow-cytometry (FCM)
PCs and PMVs were subjected to high sensitivity imaging flow-cytometry Amnis Image Stream MK II (ISX) (Amnis, America) and further analyzed by INSPIRE (v1.4.0). In brief, samples were stained with CD146 (1:100, BD, America), α-SMA (1:80, BD, America), CD140b (1:50, BD, America), NG (1:100, BD, America) and Annexin V (1:50, BD, America) for 30 min in the dark (RT). Annexin V binding buffer (BD, America) was used along with Annexin V antibody. Standard beads were added into MV samples (0.2 μm, 0.5 μm, 0.8 μm, Bangs laboratories, America) for the gate set and quantification of MVs.
Transmission electron microscopy (TEM)
TEM of MVs were measured as described [34]. In brief, after fixing with 2.5% glutaraldehyde buffer (4 °C) for 24 h, the samples were washed 3 times by 0.1 M PBS. Then MVs were post-fixed by 0.1% OsO4 for 80 min (4 °C) and dehydrated in a graded ethanol series. Next, MVs were embedded in TAAB 812 and cut into 100 nm sections, then were observed by JEM 1400 (JEOL Instruments, Japan).
Hematoxylin and Eosin (HE) staining
Rats were sacrificed gently, and the left lung was flushed with PBS formaldehyde-fixed, and finally it was embedded by paraffin and then cut into sections. After staining by HE, the sections were observed by microscopy (Leica, Germany).
Immunofluorescence (IF) of lung
FITC-BSA (9 mg/kg) (Sigma, America) was injected into anesthetized rats through the jugular vein. After being flushed with PBS, the left lung was embedded in Tissue-Tek O.C.T. Compound (Sakura, America) at − 20 °C. When the compound was frozen, it was cut into sections by freezing microtome (Leica, Germany), and the sections were stained with DAPI (Abcam, America) and observed by confocal microscopy (Leica, Germany) [34, 36].
Transendothelial electrical resistance (TER) and FITC-BSA leakage of VECs
TER and BSA leakage of VECs were detected as previously described [33]. In brief, VECs were seeded on the upper layer of Transwell (6-well, 0.4 μm, Coring, America), and VECs were added with different treatments when cells grew to full confluence (LPS: 2 μg/ml, PMV: 2 × 106/ml). TER of VECs was assessed immediately after the treatment by oltohmmetre (World Precision Inc, America), and the value of TER was recorded every 30 min. For the assessment of BSA leakage, FITC-BSA (10 μg/ml) was added into VECs immediately after the treatment, and 200 μl supernatant of the lower insert of Transwell was collected and measured every 10 min, and 200 μl DMEM-F12 medium was supplemented into the upper insert to keep the balance of the culture system in Transwell.
Angiogenesis measurement
Matrigel Matrix (Corning, America) was thawed at 4 °C and subsequently added in the µ-slides (ibidi, Germany) gently, then the µ-slides were incubated at 37 °C for 1 h. Afterward, VECs were seeded on the matrix and incubated with LPS and MVs according to the experiment design for 12 h. Then the µ-slides were labeled with calcein-AM (Thermo, America) and observed by Leica SP5 laser confocal microscopy (Germany) [37]. The tube length of angiogenesis analysis was further measured by imageJ software (v1.8.0).
Western blot
Total protein mass was extracted by RIPA buffer from either tissues or cells, and SDS-PAGE and PVDF membrane was used. After being incubated with antibodies, the membrane was observed by Odyssey Clx (Li-Cor, America). PCNA (Proliferating Cell Nuclear Antigen), AKT, p38-MAPK, JNK and ERK1/2 were purchased from CST (America), CTGF and β-actin were purchased from Abcam (America).
Migration of VECs
6-well plate (Corning, America) was used to detect the migration of VECs. Firstly, VECs were seeded at the concentration of 1 × 105 per well and cultured to full confluence. Then the medium was change to DMEM-F12, and a sterile pipette was used to draw a straight line in the middle of the well. Afterward, VECs were washed and cultured with different treatments, and were observed by microscopy (Leica, Germany) at 0 h and 24 h.
Pericyte transfection and harvest of modified PMVs
PCs were transfected with CTGF-shRNA adenovirus, CTGF-overexpressing adenovirus, and CTGF-mock adenovirus (Genechem Technology, China) according to experiments design. Briefly, corresponding adenovirus was co-incubated with PCs in the presence of Opti-MEM when cells were 70–80% confluence for 24 h (MOI = 80), then the medium was changed to exosome-free DMEM-F12 for another 24 h, and modified PMVs were harvested from the supernatant [29]. The target sequence of CTGF-shRNA was as follows:
Forward Strand: 5′-GATCCCTACCGACTGGAAGACACATTTCTCGAGAAATGTGTCTTCCAGTCGGTATTTTTGGAT-3′, Reverse Strand: 5-AGCTATCCAAAAATACCGACTGGAAGACACATTTCTCGAGAAATGTGTCTTCCAGTCGGTAGG-3′.
LC–MS/MS analysis
PMVs were harvested as described (PC-MV1, PC-MV2, PC-MV3), and smooth muscle cell-derived microvesicles (SMVs) were used as control group (SMC-MV1, SMC-MV2, SMC-MV3). MV samples were analyzed by Applied Protein Technology Biolaboratory (China) by Q Exactive mass spectrometer (Thermo Scientific, America). Briefly, samples were resuspended in SDT buffer A (100 mM Tris–HCl, 4%SDS, 1 mM DTT, and pH was adjusted to 7.6) and quantified by BCA (Dojindo, Japan). Afterward, 200 μg protein was incorporated into SDT buffer B (30 μl, 150 mM Tris–HCl, 4% SDS, 100 mM DTT, and pH was adjusted to 8.0), then DTT and the detergent were removed by UA buffer (150 mM Tris–HCl,8 M Urea, pH was adjusted to 8.0) using ultrafiltration (Microcon units, 10 kD). Afterward, samples were added with 100 μl iodoacetamide (100 mM IAA in UA buffer) and incubated in darkness (0.5 h), and digested by trypsin (4 μg, Sigma, America) in 25 mM NH4HCO3 buffer overnight (RT), and these digested peptides were collected in a filtrate. Then peptides were desalted on C18 (Empore™ SPE Cartridges C18, 2.8 ml, bed I.D. 7 mm, Sigma, America), concentrated by vacuum centrifugation and reconstituted in 0.1%formic acid [38].
The purified peptides were loaded on the reverse phase trap column (Thermo Scientific, America), which was connected to the C18-reversed phase analytical column (Thermo Scientific, America) in 1‰ formic acid buffer A, then samples were separated by gradient buffer B (1‰ formic acid and 84% acetonitrile), with a flow rate of 300 nl/min. Then the purified peptides were analyzed by mass spectrometry and the data were collected by a data-dependent method dynamically choosing precursors ions from the scan range of 300–1800 m/z, and the parameters were as follows: 3e6 automatic gain control target; 10 ms maximum inject time; 40.0 s dynamic exclusion duration; a resolution of 70,000 at m/z 200 survey scans; 2 m/z isolation width; 30 eV normalized collision energy; peptide recognition mode [38].
Database search and bioinformatic analysis
These MS/MS raw data were searched and analyzed by MaxQuant software (v1.5.3.09) for identification and quantitation information. Trypsin was used as cleavage enzyme, and the max missed cleavages were set to 2. Fixed modification was Carbamidomethyl (C), and variable modification was Oxidation (M), and the mass tolerance for precursor ions for First search and Main search was 20 ppm and 6 ppm, respectively. False discovery rate (FDR) was set to ≤ 0.01, and peptides for quantification were set as Use razor and unique peptides, and time window was 2 min. The differentially expressed (DE) proteins were identified with absolute fold change > 1.5 and p value < 0.05 [38].
The sequences of the DE proteins were searched and analyzed by the NCBI BLAST + client software (v2.2.10) and InterProScan (v5.0), and Gene Ontology (GO) annotation were mapped by BLAST2GO and plotted by R scripts. Then differentially expressed proteins were blasted against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://geneontology.org/) and mapped to pathways in KEGG. Proteomic enrichment analysis was performed based on the Fisher’ exact test, and p value < 0.05 was considered significant [38].
Statistical analysis
In the present study, experiment data were analyzed by SPSS (v20.0, IBM, America). All the data were repeated at least 3 independent experiments, and results of one representative experiment were shown. Data were presented as mean ± SD, and difference among different groups were assessed by one-way ANOVA test, and values of p < 0.05 were considered significant.