Patients with aortic dissection (AD) may present acute lung injury (ALI) that may affect the prognosis. In this study, we aim to investigate the roles and mechanism of IL-22 in the pathogenesis of AD complicated with ALI. Six hundred and twenty-one AD patients were included, and the incidence of ALI and pulmonary CT findings were analyzed. Mouse ALI model was established through AngII, and then IL-22 injection and AG490 were given. The pathological changes, infiltration of inflammatory cells, and expression of STAT3 were determined. For the in vitro experiment, cultivated pulmonary microvascular endothelial cells (PMVECs) were treated by angiotensin II (AngII), followed by treating with IL-22 and/or AG490. The expression and migration of STAT3 was determined. Flow cytometry was carried out to evaluate the apoptosis. IL-22 contributed to the expression of STAT3 in lung tissues and attenuation of ALI. IL-22 obviously inhibited the apoptosis of PMVECs mediated by AngII and downregulated the expression and intranuclear transmission of STAT3. Such phenomenon was completely inhibited upon administration of AG490, an inhibitor of JAK2. Our data showed IL-22 contributed to the inhibition of PMVEC apoptosis mediated by AngII through activating the JAK2/STAT3 signaling pathway, which may attenuate the ALI induced by AngII.
Patients with aortic dissection (AD) may present acute lung injury (ALI), and the treatment outcome is much severe than those with single AD [
Interleukin-22 (IL-22), a member of IL-10 family, is initially discovered in 2000 by Dumoutier et al. [
Six hundred and twenty-one AD patients admitted in our department from March 2008 to March 2015 were included in this study. AD was diagnosed based on the CT angiography of aorta. Besides, those with chronic pulmonary disorders, with a long-term history of hormonal therapy or medication of anti-inflammatory agents, were also excluded. The diagnosis of ALI was based on the PaO2/FiO2 of ≤300 mmHg. Written informed consent was obtained from each patient. The study protocols were approved by the Ethical Committee of Renmin Hospital of Wuhan University.
Male mice (8 weeks old), purchased from HFK Bioscience Co., Ltd (C57BL/6J, Beijing, China), were divided into four groups after the one-week adaptation, including (i) control group, fed on a normal diet; (ii) AngII group, subject to AngII (1
Rat PMVECs were purchased from BeNa Culture Collection Co., Ltd. (category number BNCC338210; Peking, China). Cells were cultured in endothelial culture medium (number 1001, Sciencell) containing 5% fetal bovine serum (FBS), 1% endothelial cell growth supplement (number 1052, Sciencell) and 1% penicillin/streptomycin solution (number 0503) in 5% CO2 at 37°C. Cells (P2–P4) cultured in ECM medium containing no FBS for 24 were divided into four groups: (i) normal control, cultured in low-serum RPMI 1640 medium containing 2% FBS; (ii) AngII group, cells cultured in low-serum RPMI 1640 medium containing 2% FBS and AngII (1
Electron microscope was performed to observe the structural changes of PMVECs. Briefly, the lung tissues obtained from the cadavers with AAD complicated with ALI were fixed using 2.5% glutaraldehyde, followed by washing with phosphate buffer (0.1 M) for 3 hrs. Afterwards, OSO4 (2%) was added and mixed for 2 hrs, followed by embedding in the Epon-Araldite. Finally, the samples were subject to staining by uranyl acetate and lead citrate. The images were observed under an H-7700 transmission electron microscope (Hitachi, Tokyo, Japan) to determine the changes of structural changes of PMVECs.
The mouse lung tissues were fixed and embedded as routinely described. The sections (4
The total protein was extracted from the mouse lung tissues and cultured PMVECs. Protein content was evaluated using the BCA commercial kit (Beyotime Biotechnology, Jiangsu, China). The transferred membrane was blocked with 10% skimmed milk for 1 h at room temperature, and then the blocked membrane was incubated with the primary antibody against STAT3 (1 : 1000; Abcam) and
Cell apoptosis was determined using flow cytometry after annexin V/PI staining. The results were analyzed using Expo32 ADC analysis software.
Quantitative data was presented as the mean ± standard error of mean. Statistical differences were analyzed using Student’s
In total, 621 cases with AD were included in this study, among which 217 (34.9%) showed concurrent ALI (Table
Clinical data of AD patients.
Variable | Overall | ALI | Non-ALI |
|
---|---|---|---|---|
|
621 (100%) | 217 (34.9%) | 404 (65.1%) | |
Age, y | 50.0 ± 9.3 | 49 ± 6.8 | 52.1 ± 11.2 | |
Male sex | 502 (80.8%) | 185 (85.3%) | 317 (78.5%) | 0.0425 |
Smoking | 309 (49.8%) | 112 (51.6%) | 197 (48.8%) | 0.5022 |
Hypertension | 573 (92.3%) | 204 (94.0%) | 369 (91.3%) | 0.2718 |
Acute | 480 (77.3%) | 209 (96.3%) | 271 (67.1%) | <0.0001 |
Type of AD complicated with ALI.
Variable | ALI (217) | Non-ALI (404) |
|
---|---|---|---|
Stanford A | 140 | 104 | |
Surgery | 118 (82.3%) | 88 (84.6%) | 1.0000 |
Interventional therapy | 0 | 0 | |
Medical management | 22 (15.7%) | 16 (15.4%) | 1.0000 |
Stanford B | 77 | 300 | |
Surgery | 6 (7.8%) | 13 (4.3%) | 0.2415 |
Interventional therapy | 54 (70.1%) | 209 (69.7%) | 1.0000 |
Medical management | 17 (22.1%) | 78 (26%) | 0.5571 |
Comparison of pulmonary CT findings in patients with AAD or normal individuals. The pulmonary markings were clear in these patients with no solid shadows or exudation.
As revealed in Figure
Electron microscope findings in the lung tissues of normal control (a) and patients with AAD complicated with ALI (b). Infiltration of macrophages was observed in the PMVECs in the patients with AAD complicated with ALI, together with karyopyknosis in various forms and chromatin margination. The images were observed under a magnification of 1500x.
According to our previous study, serum AngII showed obvious elevation in the ALI patients [
IL-22 induced obvious inhibition of ALI induced by AngII. (a) All mice showed ALI after AngII treatment, and IL-22 could decrease the incidence of ALI. (b) In the AngII group, obvious pulmonary edema was noticed in the lung tissues. (c) Presence of massive infiltration of neutrophils (MPO) and macrophages (CD68). Such phenomenon was completely reversed after IL-22 treatment. AG490 could obviously inhibit the pulmonary protective effects of IL-22. The HE images were observed under a magnification of 100x. The immunohistochemistry images were observed under a magnification of 200x.
The expression of STAT3 in the mouse lung tissues in the AngII + IL-22 group was obviously higher than that of the normal control group and AngII group, respectively, as revealed by the immunohistochemistry analysis and Western blot analysis (Figure
IL-22 contributed to the expression of STAT3 in the mouse lung tissues and was completely inhibited after treating with AG490 as revealed by immunohistochemistry (a) and Western blot analysis (b).
Our previous data indicated AngII-induced apoptosis of PMVECs was responsible for the onset of ALI [
IL-22 inhibited the PMVEC apoptosis induced by AngII. Flow cytometry revealed the apoptosis rate of PMVECs in the IL-22 group was obviously decreased compared with that of the AngII group, whereas the apoptosis rate of PMVECs in the AngII + IL-22 + AG490 group was remarkably increased compared with that of the AngII + IL-22 group.
JAK/STAT signal pathway plays crucial roles in the IL-22-mediated antiapoptosis and inflammation. In this study, Western blot analysis revealed the expression of STAT3 in the PMVECs subject to AngII + IL-22 was obviously upregulated compared with that of the AngII group (Figure
IL-22 contributed to the expression and nuclear transfer of STAT3 in PMVECs. (a) Western blot analysis indicated IL-22 contributed to the expression and nuclear transfer of STAT3; however, such phenomenon was inhibited by AG490. (b) Immunofluorescence assay indicated IL-22 contributed to the expression and nuclear transfer of STAT3, which was attenuated after interference of AG490.
AD, a severe condition causing great threats to the public health, may trigger multiple organ disorders and systemic inflammation [
The roles of AngII in the ALI were mainly featured by inducing systemic inflammation and increase of vascular leakage [
In this study, ALI mouse model was established through pumping of AngII, in which obvious edema was noticed in the lung tissues, together with massive infiltration of neutrophils and macrophages, whereas the ALI was attenuated after IL-22 treatment. As a protective factor, IL-22 has been reported to play protective roles in various cells and animal models, such as ischemia-reperfusion injury in lung and active chronic inflammation in the intestine tracts [
Electron microscope confirmed the proapoptotic changes in PMVECs in the AAD complicated with lung injury, which indicated the apoptosis of PMVECs involving in the pathogenesis of AAD complicated with ALI. Knowing the inhibitory effects of IL-22 on PMVEC apoptosis mediated by AngII, we speculated that IL-22 may play protective roles in the lung injury through inhibiting the PMVEC apoptosis induced by AngII. For the mechanism, IL-22 may bind with the receptors and act on the target cells through activating the JAK/STAT signal pathways, which subsequently induced the phosphorylation of STAT1, STAT3, and STAT5, respectively. Meanwhile, IL-22 could activate the MAPK signal pathway through inducing the phosphorylation of Erk1/2, JNK, and p38 [
After IL-22 interference, the expression of signal transducers and activators of transcriptions was obviously upregulated in the PMVECs, together with intranuclear transmission. Such phenomenon was remarkably inhibited by the AG490, a selective inhibitor of JAK kinase family. As a member of protein family involved in the cellular signal transmission, STAT3 has been reported to be participate in the cell growth, differentiation, and apoptosis [
The incidence of AD complicated with ALI is more than 30%, and many patients may present hypoxemia. Such condition may induce extended duration of respirator application and pulmonary infection, which is considered as the major cause for the AD-related mortality. Previously, a prevalence of up to 20% was reported in those complicated with ALI [
Our data indicated IL-22 may inhibit the PMVEC apoptosis induced by AngII through JAK2/STAT3 signal pathway. This finding contributes to the understanding on the roles of IL-22 in the endothelial cells. It may provide a new treatment target for the AD complicated with ALI.
Acute aortic dissection
Acute lung injury
Angiotensin II
Pulmonary microvascular endothelial cells
Interleukin-22.
The whole study was approved by the Ethical Committee of the Renmin Hospital of Wuhan University (Wuhan, China).
The content for publication was obtained from the person who participated in the study.
The authors declare that they have no conflicts of interest.
Zhiwei Wang and Zhiyong Wu designed the study. Zhiwei Wang wrote the manuscript. Wei Ren did the experiment. Zhipeng Hu, Feifeng Dai, and Jinxing Chang did the data collection. Bowen Li, Huagang Liu, and Yongle Ruan analyzed the data.
This study was supported by the National Natural Science Foundation (nos. 81570428 and 81501376) and Hubei Province Health and Family Planning Scientific Research Project (no. WJ2017M013).