Background
Acute respiratory distress syndrome (ARDS) is presented as noncardiogenic pulmonary edema-induced hypoxia caused by acute lung injury (ALI) secondary to lung excessive inflammation [
1]. An approximately 75% of ARDS is associated with sepsis and presents severe mortality and morbidity [
2]. Owing to the vast surface area of pulmonary microvascular endothelium for effective gas exchange, the pulmonary vascular endothelial cells (ECs) are vulnerable to circulating stimuli during sepsis [
3]. The pro-inflammatory mediators in circulation lead to ECs dysregulation with abnormal increases in the expression of pro-inflammatory cytokines and cellular adhesion proteins including vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectins [
4‐
6]. Excessive inflammation further impairs pulmonary microvascular integrity due to the decrease in endothelial cell–cell junction proteins (e.g. cadherin) and ECs apoptosis, resulting in pulmonary vascular permeability disruption, alveolar edema, additional immunocytes trafficking and uncontrolled alveolar inflammation [
7]. Hence, inflammation-mediated pulmonary endothelial dysfunction is considered to be the main pathogenesis of septic-ARDS. Understanding the mechanism of circulating inflammatory imbalance in pulmonary endothelial dysfunction is of crucial importance.
Compared to other lipoproteins, HDL has a critical role in maintaining the endothelial integrity due to its anti-inflammatory and anti-oxidative properties [
8]. Upon septic stresses, HDL processes the anti-inflammatory function through both neutralizing lipopolysaccharide (LPS) and alleviating ECs inflammatory responses [
9]. Septic patients exhibit a marked reduction in plasma HDL cholesterol (HDL-C) level and the low level of HDL-C is a poor prognostic factor for severe sepsis [
10‐
12]. In addition, an adverse transition of HDL to pro-inflammation was observed during acute inflammatory disorder diseases including sepsis [
13‐
15]. Previous studies indicated that the abnormal oxidation of apoA-I by the myeloperoxidase (MPO) could be one of potential causes for the adverse transition of HDL in acute phase [
13,
16]. However, the adverse remodeling of HDL in pathogenesis of septic-ARDS is large unknown due to the complexity of HDL components. It is therefore necessary to investigate the changes in HDL composition and function in septic-ARDS patients.
In this study, we, for the first time, examined the HDLs isolated from 40 septic ARDS patients (A-HDL) and matched 40 normal controls (N-HDL) through in vivo and in intro studies. In addition to the decrease in the plasma levels of HDL-C and HDL-apolipoproteins, we found significant alterations of HDL composition in ARDS patients. The further in vivo studies indicated that the treatment of A-HDL exacerbated CLP-induced mouse ALI, suggesting an adverse functional transition of A-HDL during septic-ARDS. The deleterious effects of A-HDL were associated with pulmonary endothelial deregulation, including increased endothelial permeability, aberrant expressions of cytokines and cellular adhesion proteins. These findings clearly revealed that the remodeling in HDL composition promotes septic-ARDS via endothelial dysfunction.
Methods
Subjects
All human studies were approved by the Ethics Committee of Beijing Chaoyang Hospital, Capital Medical University (Certificate No. 2018-KE-324) and the informed consents were signed by all subjects or their representatives. The cohort study enrolled adult ARDS subjects (aged from 18–85 years old) secondary to sepsis with the requirement of invasive mechanical ventilation (IMV) and without metabolic disorders including diabetes and hyperlipidemia from the medical ICU and paired adult healthy subjects (volunteers) without chronic and acute diseases from the health screening center at the Beijing Chaoyang Hospital, Capital Medical University. Septic-ARDS was defined in accordance with the Berlin definition and 2016 Sepsis 3.0 definition and the blood samples were collected within 24 h after ARDS confirmation. In total, the blood samples from 40 ARDS patients and 40 age and sex-matched healthy controls were collected (Table
1 for the clinical characteristics). We collected 12 ml of blood from ARDS patients and 6 ml of blood from control subjects, respectively. Blood was drawn into a 6-ml EDTA vacutainer and centrifuged at 3000 rpm for 10 min and the plasma was frozen at − 80 °C for further studies.
Table 1
Clinical and demographic characteristics of ARDS patients and normal subjects
Male, n (%)a | 28 (70.0) | 25 (62.5) | 0.48 |
Age (years) b | 67 (55–74) | 62 (57–64) | 0.13 |
Etiology, n (%) | | N/A | |
Pneumonia | 25 (62.5) | | |
Abdominal infection | 15 (37.5) | | |
Underlying diseases, n (%) | | N/A | |
Hypertension | 18 (45.0) | | |
Chronic respiratory disease | 2 (5.0) | | |
PaO2/FiO2 ratio, n (%) | | N/A | |
200–300 | 8 (20.0) | | |
100–200 | 20 (50.0) | | |
≦100 | 12 (30.0) | | |
CRP (mg/dl) | 12.0 (8.7–17.1) | N/A | |
PCT (ng/ml) | 2.7(0.7–18.4) | N/A | |
APACHE II score | 15 (11–24) | N/A | |
SOFA score | 7 (4–10) | N/A | |
Complications, n (%) | | N/A | |
Liver disfunction | 20 (50.0) | | |
Acute kidney injury | 7 (17.5) | | |
Treatment during ICU, n (%) | | N/A | |
Vasopressor | 24 (60.0) | | |
Parenteral nutrition | 18 (45.0) | | |
Sedative | 31 (77.5) | | |
IMV days | 16 (9–32) | N/A | |
28-day mortality, n (%) | 13 (32.5) | N/A | |
Isolation of HDL subfractions
HDL (1.063 < density < 1.21 g/ml) was isolated from plasma by discontinuous density-gradient ultracentrifugation [
17]. Plasma from 5 individuals with similar age and clinical situations (including etiology, APACHE II score and inflammatory condition) were pooled to improve the quality of isolated HDL. Mixed plasma samples were adjusted to a density of 1.3 g/ml with KBr and the plasma was carefully overlaid with normal saline after loaded to centrifuge tube. The samples were centrifuged at 350,000 g for 5 h at 4 °C and HDLs in the middle of the tubes were carefully collected by penetrating with a syringe. The lipoprotein fractions were then dialyzed against endotoxin-free phosphatebuffered saline (10 mM, PH7.4) at 4 °C for 24 h. HDLs were sterilized with 0.22 μm filter. The purity of HDLs were confirmed by the 10% SDS-PAGE electrophoresis. The concentration of HDLs were quantified through the measurement of apoA-I content by nephelometry.
Cecal ligation and puncture (CLP) septic mouse model
The animals were bred at the animal facility of Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. All animal procedures were approved by the Animal Care and Ethics Committee of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences and were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Chinese Academy of Sciences.
ApoA-I knockout (KO) mice on C57BL/6 background were obtained from The Jackson Laboratory. CLP was performed on 10-week-old mice. Briefly, mice were anesthetized by 2% sodium pentobarbital (110 mg/kg) and a 1.0–2.0 cm of midline incision was made below the diaphragm on shaved and sterilized abdomen (scrubbed with hair cream and povidone-iodine) to expose the cecum. Following a 30% ligation (light CLP) or a 50% ligation (moderate CLP), cecum was punctured twice with a 18-gauge needle and gently compressed to extrude a small amount of cecal material. The cecum was returned to the abdomen, and the muscle and skin incisions were closed with 4–0 silk suture. Sham group was similarly treated without ligation and puncture of the cecum. After the surgery, mice were resuscitated with 1 ml pre-warmed (37 ℃) phosphate-buffered saline subcutaneously. 24 h post CLP, the lung tissues were collected and subjected into further analyses.
Cell experiments
Mouse lung microvascular endothelial cells (MLECs) were isolated from C57BL/6 mice. Briefly, the lung was perfused, lavaged and cut into small pieces which were in turn digested with the enzymes dispase and collagenase A (Sigma) for 60 min at 37 °C. Following digestion, single-cell suspensions were passed through a 70-μm filter to remove debris. Endothelial cells were isolated by positive selection using Microbeads binding to CD31. Flow cytometry confirmed that 90% of cells in the final suspension are CD31-positive. Primary MLECs were maintained in endothelium cell medium (Sciencell). For HDL treatment experiments, endothelial cells were cultured in endothelium cell medium (containing 1% FBS) with HDL (50 μg/ml) or human albumins (sigma).
In vitro permeability assay
MLECs were cultured on transwell inserts (diameter: 6.5 mm, pore size: 0.4 μm, Corning). Until cells formed a monolayer, the culture medium in upper and lower compartments was changed to medium (1% FBS) with HDL (50 μg/ml) or human albumin and cells were cultured for 24 h. The fluorescent tracer, fluorescein isothiocyanate (FITC)-dextran (500 μg/ml, Sigma-Aldrich) was then added to the upper compartment. The permeability was determined by the diffused amount of FITC-dextran into the lower compartment measured by fluorescence intensity of the medium at 518 nm with excitation at 492 nm.
Oxidative modification of apoA-I
The protein bands were digested according to an in-gel digestion procedure and were subjected into liquid chromatography–mass spectrometry (LC–MS/MS) analysis performed on an LTQ linear ion trap mass spectrometer. Modification analysis was carried out by LC–MS/MS searching the data specifically against the sequence of apoA-I using the program Sequest bundled into Proteome Discoverer 1.3 (Thermo Scientific, San Jose, CA, USA). The native reference peptide (NRP) method was used for the quantification of modified peptides present in a tryptic digest by utilizing an unmodified peptide from the protein of interest that is also formed in the tryptic digestion as the internal standard. The peak areas for interesting peptides were determined and normalized to the peak areas of the reference peptides as an index of the relative amounts of these peptides [
18].
Lung lavage analyses
The bronchoalveolar lavage fluid (BALF) was collected as described previously [
19,
20]. Briefly, 1 ml saline was instilled into lung through a 20-gauge blunt-tipped needle inserted into the trachea and aspirated three times. The supernatant of BALF (400 g × 10 min) was used for the measurements of total BALF protein (Bradford) and inflammatory cytokines by Elisa kits.
Histological analyses
Lung dissected out from mice subjected into designed procedure were fixed in 4% PFA and embedded in paraffin. These paraffin embedded tissues were sliced into 5 μm-thick sections for Hematoxylin and eosin (HE) (ScyTek Laboratories). Immnunohistochemistry (IHC) was performed as a standing protocol and the antibody used was VCAM1 (CST, 39,036).
The histopathologic degree of lung injury was evaluated through a double-blind examination and scored semi-quantitatively by a scale of 0 to 4 (0, appears normal; 1, light; 2, moderate; 3, strong; 4, intense) for edema, inflammation, hemorrhage and area of structural impairment, based on 10 fields of lung parenchyma (200 × magnification). A mean score for each of the parameters was then calculated. The final lung injury score was obtained by averaging the score from the animals within each group.
Wet-to-dry lung weight ratio and Evans Blue dye leakage
The right up lobe of lung was collected to assess the lung wet-to-dry weight ratio by the gravimetric method. After the wet lung weight was measured, the lung was incubated at 60 °C for 72 h. Then, the dry lung weight was measured and the ratio of wet-to-dry weight was calculated.
To examine the alveolar microvascular leakage after CLP, mice were injected via the tail vein with Evans Blue dye (50 mg/kg). Three hours after dye injection, BALF was collected and the supernatant of remaining BALF (400g × 10 min) was used for the measurements of absorbance at 620 nm.
The Levels of Cytokines and apolipoproteins
The HDL components including apolipoproteins, serum amyloid A (SAA), paraoxonase-1 (PON1) and myeloperoxidase (MPO) were quantified by Quantitative Competitive Elisa kits from Hermes Criterion Biotechnology. The plasma levels of human apolipoproteins, PON1, MPO and mouse HDL were measured by Elisa kits from Cloud-clone. The levels of all cytokines were measured by Elisa kits from R&D Systems.
Quantitative Real-time PCR
Total RNA was extracted from mouse lungs and MLECs using TRIzol® Reagent (Invitrogen) according to the manufacturer’s manual. RNA was transcribed into cDNA using random oligonucleotide hexamers in First Strand cDNA Synthesis Kit (Invitrogen). The data were normalized to the RPL19 content and analyzed by the 2−△△Ct method relative to control groups. The primers used in qPCR: TNF-α: CAGGCGGTGCCTATGTCTC, CGATCACCCCGAAGTTCAGTAG; IL-1β: GAAATGCCACCTTTTGACAGTG, TGGATGCTCTCATCAGGACAG; MCP-1: TAAAAACCTGGATCGGAACCAAA, GCATTAGCTTCAGATTTACGGGT; IL-6: CTGCAAGAGACTTCCATCCAG, AGTGGTATAGACAGGTCTGTTGG.
Immunoblot analyses
Protein extracts prepared from tissues and cells were subjected to immunoblot analyses with antibodies against: VE-Cadherin from Thermo Fisher, VCAM1 and P-p65 from CST, ICAM1 form Abcam, P65 and GAPDH form Santa Cruz.
Statistical analysis
Data are presented as n for categorical variables, mean ± SEM (stand error of the mean) for normally distributed continuous variables, medians (25th–75th percentiles) for no-normally distributed continuous variables. To compare continuous variables, the Shapiro–Wilk test was used to test the normality of the data. Statistical comparisons between 2 groups were performed using χ2 test for categorical variables, the Mann–Whitney U test for non-normally distributed continuous variables and the two-tailed Student’s t-test for normally distributed continuous variables. Comparisons among groups were performed using 1-way or 2-way ANOVA followed by post hoc multiple comparison tests where appropriate. A p value of < 0.05 indicated significant difference between groups.
Discussion
Herein, we indicated that sepsis-induced changes of HDL quality predispose the lung to ALI/ARDS via exacerbating pulmonary endothelial dysfunction, evidenced by key findings: (1) The septic-ARDS patients with increased pro-inflammatory cytokines showed marked alterations of HDL composition including the fractions of apolipoproteins and SAA. (2) The HDL from septic-ARDS patients showed deleterious remodeling to exacerbate CLP-induced ALI without increasing the plasma level of LPS. (3) The remodeling of HDL caused direct adverse effects on pulmonary vascular endothelial cells via enhanced pro-inflammatory properties. These findings advance the pathogenesis and therapeutic perspectives of septic-ARDS.
The remodeling of HDL in ARDS patients
Since apoA-I as the major apolipoprotein in HDL mediates critical protective functions of HDL including LPS neutralization and reversal cholesterol transport (RCT) from macrophages, the dysfunction of apoA-I has a critical contribution to inflammation-associated acute and chronic pulmonary diseases [
21‐
23]. However, apoA-I can be released in alveoli by alveolar epithelial cells and macrophages to regulate lipid homeostasis and inflammation [
22‐
25]. Therefore, the observations of apoA-I dysfunction might not fully represent the functional remodeling of HDL in septic-ARDS with systemic inflammatory disorder.
Our studies showed the significant decreases in plasma levels of HDL-C and HDL-associated apolipoproteins with marked alterations in HDL composition in these patients. These observations suggest that the depletion of HDL is likely associated with the development of septic-ARDS, although the correlation between HDL level and ARDS severity failed to reach statistic significance due to the limited number of patients. Our findings are consistent with the previous studies that patients with sepsis showed a lower level of HDL than patients with trauma in ICU. These studies also indicated that the decreased level of HDL-C was an independent predictor for persistent organ failure and mortality in acute pancreatitis [
26‐
28].
In addition, excessive inflammatory deregulation also caused changes in HDL profile indicated by proteomics studies on the HDL from acute coronary syndromes (ACS) patients showed increases in apoA-IV and apoE, suggesting a critical role of HDL remodeling in acute diseases [
29,
30]. HDL isolated from uremia patients contains enriched components of apoC-III, SAA and triglycerides, which have detrimental effects on HDL function such as RCT from macrophages [
31]. In line with these observations, our data indicated the changes of apolipoprotein fractions in the HDL from septic-ARDS patients, including significant increases in apoC-III and apoE. Notably, we also observed a marked increase in the fraction of SAA, an acute-phase response protein associated with enhanced inflammation. Acute inflammation stress causes an increase of SAA fraction in HDL via displacing HDL-associated proteins (apoA-I and PON1) by circulating SAA on the HDL surface [
32,
33]. Consistently, we also found that the increase in SAA fraction was accompanied by a significant decrease in apoA-I faction in A-HDL, suggesting that the replacement of apoA-I by SAA could contribute the adverse transition of A-HDL.
The anti-oxidative and anti-inflammatory functions of HDL is largely attributed to the functions of apoA-I and PON1. ApoA-I is susceptible to oxidation mediated by MPO and such modification impairs its anti-inflammatory function [
16]. However, our analyses failed to find significant increase in oxidative level of apoA-I in A-HDL. Furthermore, MPO and PON1, as HDL-associated proteins, bind and interact with HDL by forming a complex, wherein PON1 inhibits MPO activity, while MPO inactivates PON1 [
34]. In line with similar oxidative level of apoA-I, there are no significant differences in MPO/PON1 ratio between A-HDL and N-HDL. Taken together, these observations suggest that remodeling of A-HDL is likely associated with the development of ARDS and the profound increase of SAA might have large contribution to adverse functional change of HDL.
The remodeling of HDL predispose lung to ARDS via promoting disruption of pulmonary vascular endothelial homoeostasis
The vast surface area of pulmonary microvascular endothelium for effective gas exchange makes ECs vulnerable to circulating stimuli, especially upon infectional or sterile inflammatory disorders [
3]. The disruption of pulmonary endothelial homoeostasis therefore plays a causative role for sepsis-induced ARDS [
35]. In our studies, A-HDL exposure promoted CLP-induced endothelial disruption indicated by increased lung permeability and severe alveolar inflammation, which is associated with the marked decrease of junctions protein VE-cadherin and the increase of intercellular adhesion proteins for alveolar leukocyte recruitment. These observations suggest that A-HDL aggravated endothelial dysfunction via both endothelial integrity disruption and endothelial inflammatory activation. Additionally, although the extrinsic endothelial cell apoptosis has been shown to be unregulated in ALI/ARDS [
6], we failed to observe significantly enhanced apoptosis in the lung from A-HDL treated mice, suggesting that A-HDL exposure would promote the pro-inflammatory activation of endothelial cells rather than enhancing cell apoptosis.
Upon systemic inflammatory activation, circulating pro-inflammatory mediators activate pulmonary endothelial cells, characterized by increased expressions of pro-inflammatory cytokines and cell surface adhesion proteins [
36,
37]. Herein, our in vitro studies showed that the exposure of A-HDL on primarily cultured MLECs caused marked inductions of TNF-α, IL-6 and VCAM1 as well as the reduction of VE-cadherin with increased cell permeability. These interesting findings, for the first time, provide direct evidence that the remodeling of HDL during septic-ARDS causes direct deleterious effects on pulmonary microvascular endothelial cells, suggesting the significance of HDL in crosstalk between pulmonary and systemic inflammatory regulation during ARDS. Such direct effects of HDL on endothelial cells are in line with findings in cardiovascular diseases studies showing that HDL regulates endothelial cell function via the interaction between HDL and endothelial cells [
38]. However, the interaction and downstream regulation mechanisms in such acute lung injury-induced ARDS could be different from the findings in chronic cardiovascular diseases such as atherosclerosis. Therefore, it is worth to further investigate the mechanism involved in the interaction between HDL and pulmonary endothelial cells in ARDS.
Conclusions
In conclusion, our results depicted a sepsis-induced remodeling both in HDL quantity and quality, which predisposes lung to ALI/ARDS via inducing pulmonary endothelial dysfunctions. These results, for the first, support that HDL remodeling, other than apoA-I modification, plays a critical role in the regulation of alveolar inflammation during acute systemic inflammatory disorder. These findings also advance the knowledge about the role of metabolic regulation in pathogenesis of septic-ARDS, which might share additional light to developing effective biomarkers for ARDS treatment.
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