This study was approved by the Animal Care and Use Committee of the Harbin Medical University. Male Sprague-Dawley rats (approximately 400–450 g) were purchased from the animal centre of the Second Affiliated Hospital of Harbin Medical University.
In Vitro Part
Isolation of ECFCs
We isolated and cultured ECFCs according to a previous study. First, we collected peripheral blood and isolated mononuclear cells using density-gradient centrifugation with Ficoll-Plaque Plus (Amersham Pharmacia Biotech, Uppsala, Sweden). The mononuclear cells were cultured with endothelial growth medium-2 (containing 2% foetal bovine serum). The mononuclear cells were cultured in six-well plates, which were coated with human fibronectin at 37 °C for 21 days. After 21 days, the adherent cells were harvested for further characterization.
Characterization Of ECFCs
The cells were identified according to the results of our previous study. Approximately 24 × 104 cells/well were incubated with fluorescein isothiocyanate (FITC)-conjugated Ulex europaeus agglutinin-1 (50 µg/ml) (UEA-1, Sigma-Aldrich, Saint Louis, USA) and DiI-acetyl-low-density lipoprotein (LDL) (30 µg/ml) (Invitrogen, Carlsbad, USA). After incubation with UEA and LDL, the mononuclear cells were examined using fluorescence confocal microscopy. The mononuclear cells with dual-positive staining for UEA-1 and acetyl-LDL were defined as endothelial progenitor cells. The cells were also identified with staining for vascular endothelial growth factor receptor (VEGFR) 2 (Abcam, Cambridge, UK) and CD34 (Santa Cruz Biotechnology, Santa Cruz, USA) using a fluorescence microscope. The mononuclear cells with double-positive staining for VEGFR-2 and CD-34 were also identified as endothelial progenitor cells. Based on these results, the cells were further analysed with FITC-labelled CD14 and PE-labelled CD45 antibodies using flow cytometry. The endothelial progenitor cells with double negative staining of CD14 and CD45 were identified as ECFCs. For analysis of the mechanism of ECFCs in lung injury, ECFCs were preincubated with N5-(1-iminoethyl)-l-ornithine (L-NIO, 10 µM, Santa Cruz Biotechnology) to observe the function of the ECFCs.
Cell Proliferation Assay
The cellular viability and proliferation of ECFCs were judged by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Approximately 6 × 103 ECFCs/well (pretreated with or without L-NIO) were plated in 96-well plates in EGM-2 medium. After incubation for 24 hours, all the ECFCs were incubated in EBM-2 medium and 1% FBS without serum for 12 hours. The ECFCs were then cultured in EBM-2, 1% FBS and VEGF (100 ng/mL). After 24 hours, MTT (5 ng/ml) was added, and the ECFCs were incubated for 4 hours at 37℃. Dimethylsulfoxide (150 µl) was injected into the plates, and the plates were further incubated for 10 min. The absorbance of the cells was investigated using Multiskan EX (Thermo, Finland) at 540 nm.
The Expression Of eNOS In ECFCs
The ECFCs treated with or without L-NIO were harvested, and the total protein of the ECFCs was extracted. The protein expression in ECFCs was detected by Western blots to investigate the effect of L-NIO on the expression of ECFCs.
In Vivo Part
Rat CPB model
Thirty-two male Sprague-Dawley rats (400–450 g), obtained from the animal centre of the Second Affiliated Hospital Harbin Medical University, were randomized into 4 groups: the sham group, CPB group, EPC group and EPC/L group. The rats in the sham group only received anaesthesia and tracheal intubation. The cells were pre-treated with L-NIO. Briefly, the rats were anaesthetized with 3% pentobarbital sodium (30 mg/kg) intraperitoneally. After anaesthesia, all rats were intubated and ventilated (Model 683, Harvard Apparatus, Boston, USA). The respiratory parameters were a tidal volume (Vt) of 10 ml/kg and a respiratory rate (RR) of 50 breaths/min. The fraction of inspired oxygen (FiO2) and positive end-expiratory pressure (PEEP) were set at 50% and 2 cmH2O, respectively, and the inspiratory expiratory ratio was 1:1.
After heparinization (500 IU/kg heparin), under local analgesia, 18 G and 16 G catheters were inserted into the right carotid artery and right femoral vein, respectively, to inflow and outflow the blood. Moreover, a 22 G catheter was inserted into the right femoral artery to monitor and analyse the blood sample. The CPB circuit was constructed by a 20 ml venous reservoir, roller pump (Cole Parmer instrument company, Chicago, USA) and membrane oxygenator (MeicroPort, Dongguan, Guangdong, China). Before CPB, the circuit was primed with 0.2 ml heparin, 11 ml of hydroxyethyl starch solution and 0.5 ml 7% sodium bicarbonate solution. During the experiment, the rectal temperature was monitored and maintained within 36–38 ℃ by a heat blanket. The flow rate was gradually adjusted to 100 ml/kg body weight/min and maintained for 60 min. During CPB, the mean arterial pressure was maintained within the range of 60 to 80 mmHg using the continuous injection of adrenaline. The anesthesia was maintained with 3% pentobarbital sodium (10 mg/kg) and rocuronium (0.6 mg/kg) for a 1-hour interval. After 60 min of CPB, the outflow cannula was withdrawn, and the right femoral vein was ligated. The remaining priming solution was continuously infused when the haemodynamics were stable, and the inflow catheter was withdrawn. Immediately after withdrawal of the catheter, the rats in the sham and CPB groups were intravenously injected with 1 ml of PBS, and the rats in the ECFC and ECFC/L groups were intravenously injected with ECFCs or ECFCs pretreated with L-NIO (approximately 106 cells in 1 ml of PBS) . To prevent infection, 2000 U/kg penicillin was administered, and incisions were sutured. All rats were extubated when they recovered spontaneous breathing for 24 hours. All rats were sacrificed with an overdose of anaesthetics at 24 hours after ventilation. In this study, we enrolled 10, 9 and 9 rats in the CPB, EPC and EPC/L groups, respectively, to achieve 8 rats in each group.
ECFCs And Alveolar-capillary Permeability
The arterial blood was analysed pre-CPB and at 24-hour after CPB using a Bayer Rapidlab 348 (Bayer Diagnostics, Germany). The PaO2/FiO2 ratio was calculated to evaluate the effect of ECFCs on lung gas exchange function.
Moreover, part of the lung tissue from the right upper lung lobes was harvested. The lung tissues were weighed and dried at 60 °C for 48 hours and then weighed again. The wet/dry weight (W/D) was calculated to observe the effect of ECFCs on alveolar-capillary permeability. Moreover, the protein levels in BALF were also tested.
Histopathologic Injury Evaluation
The lung tissue from the right lower lobe was collected to estimate histological changes. Lung tissue fixed with 4% paraformaldehyde was embedded in paraffin. The lung tissue was cut into 4-µm sections and stained with haematoxylin and eosin. Two independent pathologists were blinded and employed to evaluate lung histological injury with light microscopy.
The lung injury analysis was performed by two pathologists who did not participate in this study. Briefly, the pathology indexes included alveolar congestion, lung oedema, haemorrhage, infiltration of neutrophils into the airspace/vessel wall and alveolar wall thickness. The scoring was as follows: lung hemorrhage (0 = no hemorrhage, 1 = mild hemorrhage, 2 = severe hemorrhage); pulmonary interstitial edema (0 = no edema, 1 = mild edema, and 2 = severe edema); pneumocyte hyperplasia (0 = no alveolar wall thickening, 1 = mild alveolar wall thickening, 2 = severe alveolar wall thickening, and 3 = severe alveolar wall thickening with > 50% pulmonary consolidation); and infiltration of inflammatory cells (0 = no inflammatory cell infiltration, 1 = mild inflammatory cell infiltration, 2 = moderate and extensive inflammatory cell infiltration, and 3 = severe inflammatory cell infiltration). The lung injury score was I (0–2 score), II (3–6 score), III (7–8 score) or IV (9–10 score).
ECFCs And Local And Systemic Inflammation
The right bronchi were blocked using an artery clamp. Sterile saline (15 ml/kg) at 4 °C was injected into the left lung via the left bronchi and was withdrawn 5 times. After 5 withdrawals, the bronchoalveolar lavage fluid (BALF) was collected and centrifuged at 4 °C and 1,000 g for 15 min, and then the supernatant was collected and stored at -80 °C. The peripheral blood was collected pre-CPB and at 24 hours after CPB. The blood was centrifuged at 4 °C and 1,500 g for 10 min, and the serum was collected and stored at -80 °C. The cytokines TNF-α, IL-1β, IL-6, and IL-10 were detected in the BALF and serum with the corresponding ELISA kits (Wuhan Boster Bio-Engineering Limited Company, Wuhan, China).
Moreover, the number of neutrophils and the levels of elastase in BALF were also detected.
Tracking Of ECFCs In Lung Tissue
To observe the distribution of ECFCs in lung tissue, approximately 1 × 106 ECFCs (with or without pre-treated L-NIO) labelled with acetyl-LDL were injected into rats of the ECFC and ECFC/L groups. After 24 hours of CPB, the lung tissue was harvested, and ECFC tracking was performed by fluorescence microscopy. A slice of lung tissue was prepared according to the histological analysis method. The pulmonary tissue slices were deparaffinized and stained with 4,6-diamidino-2-phenylindole (DAPI) to stain the cell nuclei. The ECFCs in lung tissues were visualized by fluorescence confocal microscopy at a wavelength of 555 nm (acetyl-LDL).
Apoptosis in the lung tissue was investigated by TUNEL staining with an Apoptosis Assay kit (Roche, Mannheim, Germany). Briefly, the lung tissue slices were immersed in proteinase K at 37 °C for 30 min. The slices were washed twice with PBS. Then, the slides were incubated in the TUNEL reaction mixture (TdT and fluorochrome-conjugated dUTP) for 60 min in a dark chamber at 37 °C. After washing twice, the slides were further incubated with 1 µg/ml 4,6-diamidino-2-phenylindole for 30 min.
The slides were covered with 0.3% H2O2 to inhibit endogenous peroxidase activity, incubated with extra-avidin peroxidase and then immersed in diaminobenzidine solution. The nuclei that were stained brown were judged as apoptotic cells. In this study, apoptosis of the endothelium and epithelium was identified by two pathologists who analysed histological injury. The apoptosis index was calculated by the ratio of positive apoptotic cells to total cells in a random field from all slides.
First, the protein was extracted, and the protein levels were calculated with the Bradford assay. An equivalent protein volume of every sample was injected into the gel. After electrophoresis, the protein was transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% milk for 30 min and incubated with primary antibody [Gelsolin, Bax, Bcl-2, cleaved caspase-3, phosphorylated myosin light chain (MLC) (Sigma Aldrich, St. Louis, Missouri, USA), and phosphorylated NF-κB (Santa Cruz Biotechnology, CA, USA)] overnight at 4 °C. After washing 3 times with PBS, the membrane was incubated with secondary antibody (Santa Cruz Biotechnology). After reaction with horseradish peroxidase, the bands were visualized with enhanced chemiluminescence.
The primary outcome of this study is the PaO2/FiO2 after 24 hours of CPB. In the preliminary study of 5 rats, the PaO2/FiO2 at 24 hours post-CPB was 240 ± 33. The sample size was calculated using PASS 11. Eight rats were needed in each group to detect an increase of 30 in the PaO2/FiO2 with a power of 0.9 and α of 0.05. All the data were normally distributed and are presented as the mean (SD). The data were analysed by one-way analysis of variance and an unpaired t test. All data were analysed using IBM SPSS Statistics 19.0 (SPSS, Chicago, IL, USA). A two-tailed p-value of < 0.05 was considered statistically significant.