Lung ischemia-reperfusion injury (LIRI) usually presents with immediate impairment in lung function after transplantation. Lung ischemia-reperfusion injury is usually accompanied by the rapid development of pulmonary edema, increased pulmonary vascular resistance, and decreased airway compliance. Lung ischemia-reperfusion injury is also the main reason for primary graft dysfunction (PGD), which increases early mortality of lung transplantations. Great efforts have been made to alleviate LIRI, including hypothermic preservation, optimized preservation solutions, such as low potassium dextran solution (LPD), controlled perfusion, protective ventilation, surfactant supplementation, and therapeutic gas application (carbon monoxide, hydrogen sulfide, and hydrogen [H2]).1
Although many methods have improved donor quality, LIRI still exists as an important prognostic factor. Microvascular disturbance still occurs because of oxidation stress, inflammation, apoptosis, and sodium pump inactivation. These issues eventually result in early graft dysfunction and bronchiolitis obliterans syndrome.2,3 Thus, it is critical to find new methods or to optimize existing strategies.
Hydrogen is a small molecule with reducibility. Through its antioxidant, anti-inflammatory and antiapoptotic properties, H2 could reduce the lung injury caused by hyperoxia, mechanical ventilation, lipopolysaccharide (LPS), and other factors.4-6 We have shown that 2% H2 could exert anti-inflammatory, antioxidative, and antiapoptotic effects on lung grafts from brain dead donors.7 Hydrogen inflation during the cold ischemia period could also reduce the inflammatory response, oxidative stress and apoptosis, and improve the function of the lung graft.8,9 The above studies illustrated the protective effect of H2 and its probable mechanism for lung donors in animals. However, the specific mechanism by which H2 exerts lung protection at the cellular level has not been clarified. Further in vitro cell experiments are required.
Pulmonary microvascular endothelial cells (PMVECs) are important components of the pulmonary microvasculature and endothelial barrier. Pulmonary microvascular endothelial cells play an important role in local hemoperfusion, oxygen (O2) supply and LIRI. They are sensitive to anoxia and reoxygenation. Therefore, an anoxia and reoxygenation cellular model with PMVECs could simulate the process of IRI. An in vitro experiment can rule out the complicated in vivo environment.10 In this study, we intended to explore the protective and anti-inflammatory effects of H2 on lung grafts in the cold ischemia phase at the cell level through an in vitro lung transplantation model of PMVECs.
MATERIALS AND METHODS
The Culture and Identification of PMVECs
All protocols were approved by the Institutional Animal Care and Use Committee of Harbin Medical University. Pathogen-free Sprague-Dawley male rats weighing 30 to 50 g were anesthetized by intraperitoneal injection with sodium pentobarbital (60 mg/kg). Whole body heparinization was conducted by heparin sodium intraperitoneal injection (1000 u per animal).
Rat PMVECs were isolated using the method described by Chen et al11 and modified by Li et al.12 Briefly, the rats were euthanized by exsanguination. Then, the chest was opened, and the lungs were removed by sterile techniques. The visceral pleura was removed under a stereomicroscope (Wanheng, China). Peripheral lung tissue was cut into small pieces (< 1 mm3) in medium 199 (M199; Gibco, Carlsbad, CA) containing 20% fetal bovine serum. The large vessels were wiped off. Then, the fragments were placed in a 25 cm2 culture flask upside down in a 37°C, 5% CO2 incubator for 2 hours. Appropriate M199 supplemented with 20% fetal bovine serum, endothelial cell growth supplement (50 μg/mL), heparin (90 U/mL), and penicillin-streptomycin (100 U/mL) were added into the culture flask. After 60-hour incubation, the explants were removed, and the medium was replaced. Pulmonary microvascular endothelial cells were identified according to the results of immunocytochemistry staining of CD31 and lectin binding. Because CD31 is a specific marker of endothelial cells, and lectin binding can distinguish macrovascular and microvascular endothelial cells.13,14
The in vitro model in the present study was modified from the hypoxia/reoxygenation model of Tan et al.10 Cultured PMVECs with equal viability seeded on culture dishes under the same culture conditions were randomly divided into 4 groups: blank, control, O2, and H2 groups.
The cells in the blank group were normal cells cultured at 37°C in 5% CO2 atmosphere without treatment. In other groups, the PMVECs were put into a sealed, self-made incubator that was preaerated with 95% O2 and 5% CO2 at 1 L/min for 2 hours. The medium in the culture dishes was replaced with 4°C sterile LPD solution (pH, 7.2-7.4), which was preequilibrated with 95% O2 and 5% CO2. Then, the PMVECs were put into 4°C sealed incubators for 4 hours to simulate the cold storage period (CSP). The sealed incubator in the control group was filled with room air and aerated with no gases. The O2 group was aerated with 40% O2 and 60% N2, and the H2 group was aerated with 3% H2, 40% O2, and 57% N2 for 4 hours. When the concentration of O2 or H2 in the self-made incubator reached saturation, all the aeration pipes were occluded. The mixed gases in the incubators were replaced every 20 minutes. Then, the incubators were kept at room temperature and sealed for 1 hour to simulate the transplantation period (TP). After that, the LPD solution was replaced with 37°C sterile preheated M199 culture medium (pH, 7.2-7.4) to simulate the reperfusion period (RP). In this period, the incubators were continuously aerated with 40% O2, 55% N2, and 5% CO2 for 4 hours. The gas concentration in the incubators was monitored by a gas analyzer (S/N 32590; DATEX Ohmeda, Finland).
Detection of the Extracellular pH During Simulated Lung Transplantation
The extracellular pH in the LPD solution and culture medium were detected by a pH meter (STARTER2100, Ohaus, NJ) at CSP 0 hour, CSP 4 hours, TP 1 hour, and RP 4 hours, which represent the levels of cell hypoxia.
Measurement of Inflammatory Cytokines During Simulated Lung Transplantation
The LPD solution and the culture medium were obtained and centrifuged at 1000g (Heraeus, Germany) for 10 minutes at 4°C. The concentration of inflammatory cytokines including interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-10 in the supernatant were assayed according to the protocols of the ELISA assay kits (R&D Systems, Minneapolis, MN).
Measurement of ICAM-1, p38 Phosphorylation Level, and NF-κB Activity by Western Blot After Simulated Reperfusion
At RP 4 hours, the cells were lysed and centrifuged at 14 000g for 10 minutes at 4°C (Heraeus, Germany). Then, the supernatant with soluble protein was obtained. Nuclear and cytoplasmic proteins were extracted by a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China) according to the instructions for detecting the activation of NF-κB. Protein samples were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, the proteins were transferred onto polyvinylidene fluoride membranes. After blocking with skimmed milk, the membrane was washed 3 times in tris-buffered saline with tween 20 (TBST) for 5 minutes. The blots were incubated with primary antibodies (CST, USA) to ICAM-1, phospho-NF-κB p65 (p-p65), inhibitor of NF-κB (IκBα), phospho-p38 (p-p38), total p38 (t-p38), and β-actin overnight at room temperature. The membrane was washed with TBST 3 times every 5 minutes. Then, the membrane was incubated with secondary antibody (Jackson, USA) for 2 hours at room temperature. The blots were exposed to enhanced chemiluminescence and quantified by ImageJ 1.34s software. The results were presented as the ratio of target proteins to internal controls (β-actin).
Observation of Morphological Structure by Transmission Electron Microscopy After Simulated Reperfusion
At RP 4 hours, the adherent PMVECs were resuspended into a single-cell suspension. Then, the single-cell suspension was centrifuged at 1000g to get the cell pellet. After being submerged in 2% glutaraldehyde and postfixed in 1% buffered osmium tetroxide, the cell pellet was embedded in epoxy resin. Then, the cell pellet was cut into sections 40 to 50 nm thick. The ultrathin sections were stained with uranyl acetate and lead citrate. The morphological structure and characteristics were observed under a transmission electron microscopy (Hitachi H-7650, Hitachi, Tokyo, Japan). The mitochondrial disruption was evaluated by Flameng score (0, normal mitochondrial structure, intact particles; 1, normal mitochondrial structure with missing particles; 2, swollen mitochondrial, transparent, and clear matrix; 3, ruptured cristae, transparent mitochondrial matrix, and condensed mitochondrial; 4, ruptured mitochondrial inner and outer membrane, disrupted structure).14
Detection of Cell Apoptosis and Survival Rate by Flow Cytometry After Simulated Reperfusion
At RP 4 hours, a single-cell suspension was prepared. Cells were stained using an Annexin V-FITC kit (Beyotime, China) according to the manufacturer’s instructions. Data acquisition and analysis were performed by an FAC Scan flow cytometer (BD FACSCanto II, USA) using Cell Quest software. Annexin V-positive but PI-negative cells were identified as early apoptotic cells, Annexin V and PI-positive cells were identified as late apoptotic cells. Annexin V-negative and PI-negative cells were identified as survival cells.
All statistical analyses were performed with SPSS 20.0 statistical software. The extracellular pH, ELISA results and Western blot optical density values were analyzed by 1-way analysis of variance (ANOVA), and the differences between the 2 groups were analyzed with the Student-Newman-Keuls test. Repeated measurement data were analyzed by repeated measurement ANOVA. Flameng score was analyzed by the Kruskal-Wallis 1-way ANOVA. The data were expressed as the mean ± standard deviation or medians (interquartile ranges) based on their distributions. All experiments were repeated at least 3 times. P value less than 0.05 was considered statistically significant.
Identification of PMVECs
After explant culture for 60 hours, a mass of PMVECs migrated from the tissue under an inverted microscope (Figure 1A). The confluent PMVECs displayed a cobblestone appearance and microvascular structures (Figure 1B). The CD31 antigen expression was positive in PMVECs, which showed green fluorescence detected by CD31 immunofluorescence staining, and microvascular structures were observed under fluorescence microscope (Figure 1C). Cell nucleus 4'6-diamidino-2-phenylindole (DAPI) staining merged with FITC-conjugated lectin was observed under fluorescence microscope. The nucleus showed blue fluorescence, and the cytoplasm showed green fluorescence (Figure 1D). It was indicated that the isolated cells were PMVECs.
The Changes in Extracellular pH During Simulated Lung Transplantation
There was no significant difference in extracellular pH at CSP 0 hour. The pH values at CSP 4 hours in the control group (6.37 ± 0.10) were significantly lower than those in the blank group (7.31 ± 0.05) (P < 0.05). The pH values in the O2 (7.17 ± 0.05) and H2 groups (7.19 ± 0.08) were significantly lower than the blank group and higher than the control group (P < 0.05) at CSP 4 hours. The extracellular pH in the H2 group was significantly higher than the O2 group (P < 0.05). The trends for extracellular pH at TP 1 hour and RP 4 hours were consistent with CSP 4 hours. In addition, the pH was decreased across CSP and TP and increased significantly at RP 4 hours (Table 1).
The Level of Inflammatory Factors in the Extracellular Solution During Simulated Lung Transplantation
At CSP 4 hours, the level of IL-6 in the control group (86 ± 8 pg/mL) was significantly higher than that in the blank group (34 ± 3 pg/mL) (P < 0.05). The levels of IL-6 in the O2 (69 ± 4 pg/mL) and H2 groups (60 ± 4 pg/mL) were significantly higher than the blank group and lower than the control group (P < 0.05). The level of IL-6 in the H2 group was significantly lower than the O2 group (P < 0.05). The trend for TNF-α levels was consistent with IL-6. The trend for IL-10 levels was opposite to IL-6 and TNF-α. The IL-6, TNF-α and IL-10 expressions at TP 1 hour and RP 4 hours showed consistent trends with that at CSP 4 hours. In addition, the level of IL-6 and TNF-α increased gradually with time. The level of IL-10 had a downward trend (Table 2).
The Protein Expressions of ICAM-1, p38 Phosphorylation, and NF-κB Activation After Simulated Reperfusion
The ICAM-1 protein expression in the control group was higher than that in the blank group. The ICAM-1 expressions in the O2 and H2 groups were higher than those in the blank group and lower than those in the control group. The ICAM-1 expression in the H2 group was lower than that in the O2 group (Figures 2A-B). The t-p38 protein expression was not significantly different among groups. However, the p38 phosphorylation level and nuclear p65 were consistent with ICAM-1 (Figures 3A-B and Figures 4A-B). The expression of cytoplasmic IκBα was the opposite of ICAM-1(Figures 5A-B).
The Morphological Changes to PMVECs Under Electron Microscope After Simulated Reperfusion
The PMVECs in the blank group had normal cell structures (Figure 6A). The PMVECs in the control group had many lipid droplets and vacuoles, mitochondrial edema, matrix concentration, cristae rupture and reduction, and mitochondrial membrane rupture (Figure 6B). These morphological changes were alleviated in the O2 and H2 group contrast to the control group (Figure 6C). In the H2 group, these morphological changes were lighter than the O2 group (Figure 6D). The Flameng score of mitochondria in the control group (3 [2 to 4]) was higher than that in the blank group (1 [0 to 1]). The Flameng scores in the O2 (2 [1 to 3]) and H2 (1 [0 to 2]) groups were higher than those in the blank group and lower than those in the control group. The Flameng score in the H2 group was lower than that in the O2 group (Figure 6E).
Apoptosis Detected by Flow Cytometry After Simulated Reperfusion
After reperfusion, the percentage of cell apoptosis in the control group (12.9 ± 0.7) was significantly higher than that in the blank group (4.4 ± 0.5). The early apoptosis in the O2 (9.7 ± 0.4) and H2 groups (5.6 ± 0.6) was significantly decreased compared to that in the blank group but increased compared to that in the control group. The cell apoptosis in the H2 group was significantly lower than that in the O2 group. Late apoptosis was consistent with early apoptosis. Survival rate was opposite to late and early apoptosis (Figures 7A-B).
This study shows that aeration with O2 and 3% H2 during cold storage could protect the PMVECs in a model of PMVECs that simulates lung transplantation. With the application of O2 or the mixed gases of O2 and H2 in this experiment, the decrease in extracellular pH was inhibited, the secretions of proinflammatory cytokines were restrained, and the secretion of anti-inflammatory cytokine IL-10 was promoted, such that PMVEC apoptosis was decreased. In parallel, PMVECs cellular and mitochondrial damage were alleviated as observed by an electron microscope. H2 also inhibited the phosphorylation of p38 MAPK and the activation of the NF-κB pathway. Interestingly, the O2 group were consistent with the H2 group. Furthermore, the overall effect of the compound administration of H2 and O2 was more significant than 40% O2 alone. This beneficial effect was consistent with our previous in vivo studies.7-9
Because PMVECs are sensible for hypoxia and reoxygenation during IR and they are important targets for inflammatory factors and reactive oxide species (ROS) which leading to functional impairment. We used PMVECs to establish the in vitro model of transplanted lungs. The importance of microvascular endothelial cell injury is clinically shown by the reports that their impairment plays a vital role in the development of PGD and obstructive bronchiolitis syndrome.3,15
The backbone in the setup of the present study is that H2 can prevent LIRI by anti-inflammatory ability. As a small molecule, with a concentration gradient, H2 can directly permeate the cell membrane barrier, quickly act on the target, and eventually discharge from the lungs or metabolize into nontoxic water. Based on in vitro studies, low concentrations of H2 (< 4%) can protect against lung injury induced by hyperoxia, mechanical ventilation, LPS systemic inflammatory response, oxidative stress, or mechanical stimulation.4-6,16 In our previous studies, we also found that mechanical ventilation with 2% H2 or 3% H2 inflation during the cold ischemia phase had protective effects on lung grafts by anti-inflammatory, antioxidative and antiapoptosis properties.7,9 In the present study, the inflammatory response, cell apoptosis and cellular damage was prevented by 3% H2 administration. Although, under normal conditions, there is a risk of combustion and explosion when the H2 concentration is greater than 4%,17 the high concentration of H2 may have a better therapeutic effect. Sun et al18 claimed that 67% H2 could protect mouse hearts against ischemia/reperfusion injury. The effect of different H2 concentrations on hypoxia/reoxygenation injury in PMVECs needs to be further explored.
Interestingly, we found that 40% O2 had consistent effect with 3% H2. The possible reason is that O2 is the basal substance in cellular metabolism, it can improve the hypoxia of PMVECs. In a hypoxic state, aerobic metabolism is inhibited, which results in anaerobic metabolism and accumulation of toxic substances, including lactic acid, ROS, nitric oxide, and others. The hypoxic state causes a lack of energy supply, cell membrane lipid peroxidation and structural damage, which result in cell function impairment or even cell death.15 Previous studies found that O2 supply in the cold ischemia phase could ensure the aerobic metabolism of the lung donor and improve lung grafts quality.19,20 As a result, ATP content is increased, and the accumulation of anaerobic metabolites and ROS is inhibited.
In the present study, 3% H2 decreased the release of proinflammatory mediators. IL-6 and TNF-α are the major proinflammatory cytokines that induce lung injury, which play an important role in the cascade effect of inflammatory response. ICAM-1 is a key molecule involved in cascade amplification of lung injury and is also a lung vascular endothelial cell injury activation marker.21,22 IL-10 could have anti-inflammatory effects, which would inhibit proinflammatory cytokines.23,24 In previous studies, H2 could inhibit the release of proinflammatory mediators, such as ICAM-1, TNF-α, and IL-6 and promote the production of IL-10 to improve the lung grafts quality.7,25,26 Our previous study also found that 2% H2 inhalation could reduce the expression of proinflammatory cytokines to exert protective effect on lung grafts from brain-dead donors. The results in the present study are accordant with previous studies.
NF-κB is mainly involved in the expression of genes related to cell defense reactions and signal transduction of proinflammatory cytokines.27 Hydrogen inhibits the activation of NF-κB and reduces the secretion and release of downstream inflammatory cytokines.28-30 p38 MAPK is a member of the MAPK family that primarily exerts its regulatory effect on transcription factors through phosphorylation. Its inhibitors could decrease the accumulation of inflammatory cells and TNF-α release.31,32 Previous studies confirmed that H2 inhibited the activation of p38 MAPK, and then decreased the downstream expression of proinflammatory cytokines and neutrophil infiltration, thereby reduced LPS-induced acute lung injury.16 In addition, several studies confirmed that p38 MAPK regulated NF-κB activation by classical pathways.33–35 The inhibition of phosphorylation of p38 MAPK could block NF-κB activation, then restrain inflammatory response and apoptosis.34 The NF-κB and p38 MAPK activation was inhibited by H2 in the present study which was consistent with previous studies. This indicates that H2 might reduce the expression of TNF-α, IL-6 and ICAM-1 to exert an anti-inflammatory effect by directly inhibiting the phosphorylation of p38 MAPK or indirectly blocking the NF-κB pathway through inhibiting p38 MAPK phosphorylation.
However, this study has some limitations. (1) The in vitro cell model cannot completely simulate the complicated in vivo environment and physiological and pathological reactions of the transplant process. (2) The present study didn’t determine which of p38 MAPK or NF-κB pathway plays a role in H2 protective effect. (3) We did not evaluate impacts of pulmonary distension caused by mechanical ventilation and shear stress caused by perfusion on PMVECs. (4) In future studies, we should prolong the ventilation and observation time after reperfusion to explore the protective effect of H2 and the interaction with time. (5) Detailed protective mechanisms of H2 including antiapoptosis, antioxidation, antimitochondrial damage should be illuminated by Lung Transplantation Model of PMVECs.
In conclusion, 3% H2 and 40% O2 administration during the CSP improves the hypoxic state and alleviates the inflammatory response in a lung transplantation model of PMVECs. In addition, 40% O2 only shows consistent effect. The anti-inflammatory effect may be achieved by inhibiting the secretion of proinflammatory cytokines, such as TNF-α, IL-6 and ICAM-1, and by promoting the secretion of anti-inflammatory factors such as IL-10 by inhibiting the activation of the p38 MAPK pathway or the NF-κB pathway. Although both p38 MAPK and NF-κB are critical modulators of inflammation, further study is required to explore the detailed relationship between p38 MAPK and NF-κB pathways.
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