key: cord-0756297-tvpakql0
authors: Li, Jun; Qi, Zhijiang; Li, Dongxiao; Huang, Xiao; Qi, Boyang; Feng, Jiali; Qu, Jianyu; Wang, Xiaozhi
title: Alveolar epithelial glycocalyx shedding aggravates the epithelial barrier and disrupts epithelial tight junctions in acute respiratory distress syndrome
date: 2020-11-24
journal: Biomed Pharmacother
DOI: 10.1016/j.biopha.2020.111026
sha: 947916942b7f742d1d49e57377d53b4a4757a858
doc_id: 756297
cord_uid: tvpakql0

The main pathophysiological mechanism of acute respiratory distress syndrome (ARDS) invovles the increase in alveolar barrier permeability that is primarily caused by epithelial glycocalyx and tight junction (TJ) protein destruction. This study was performed to explore the effects of the alveolar epithelial glycocalyx on the epithelial barrier, specifically on TJ proteins, in ARDS. We used C57BL/6 mice and human lung epithelial cell models of lipopolysaccharide (LPS)-induced ARDS. Changes in alveolar permeability were evaluated via pulmonary histopathology analysis and by measuring the wet/dry weight ratio of the lungs. Degradation of heparan sulfate (HS), an important component of the epithelial glycocalyx, and alterations in levels of the epithelial TJ proteins (occludin, zonula occludens 1, and claudin 4) were assessed via ELISA, immunofluorescence analysis, and western blotting analysis. Real-time quantitative polymerase chain reaction was used to detect the mRNA of the TJ protein. Changes in glycocalyx and TJ ultrastructures in alveolar epithelial cells were evaluated through electron microscopy. In vivo and in vitro, LPS increased the alveolar permeability and led to HS degradation and TJ damage. After LPS stimulation, the expression of the HS-degrading enzyme heparanase (HPA) in the alveolar epithelial cells was increased. The HPA inhibitor N-desulfated/re-N-acetylated heparin alleviated LPS-induced HS degradation and reduced TJ damage. In vitro, recombinant HPA reduced the expression of the TJ protein zonula occludens-1 (ZO-1) and inhibited its mRNA expression in the alveolar epithelial cells. Taken together, our results demonstrate that shedding of the alveolar epithelial glycocalyx aggravates the epithelial barrier and damages epithelial TJ proteins in ARDS, with the underlying mechanism involving the effect of HPA on ZO-1.

The alveolar epithelial glycocalyx was discovered approximately 50 years ago and may represent an important component of the alveolar barrier [12, 13] . It is a carbohydrate-rich layer lining the pulmonary epithelium, composed primarily of glycosaminoglycans (mainly heparan sulfate (HS) and chondroitin sulfate) and proteoglycans. HS is critical for pulmonary hemostasis, maintaining the parenchymal structure, and facilitating cellular signaling [14, 15] . Pathological degradation of glycocalyx HS causes alveolar damage and multisystem organ failure including the development of ARDS [15] . HPA is an endo-β-D-glucuronidase, and is the only enzyme that degrades HS side chains at specific intrachain sites in mammals and selectively sheds HS from the glycocalyx [16] . Intravenous injection of heparinase III, an HS-specific bacterial glucuronidase, rapidly decreases pulmonary endothelial glycocalyx layer thickness in wild-type mice [16, 17] . Notably, the non-anticoagulant N-desulfated/re-N-acetylated heparin (NAH) inhibits HPA in mice [18] . Our previous studies indicated that intravenous injection NAH reduced lung endothelium HS shedding and alleviated extrapulmonary ARDS development in rat models [18] .

TJ is composed of transmembrane proteins such as occludins, claudins, junctional adhesion molecules, and cytoplasmic proteins such as zonula occludens (ZO)-1, ZO-2, and ZO-3 [19, 20] . Occludin binds directly to ZO-1, and ZO-1 binds to the intracellular cytoskeletal proteins, suggesting that ZO acts as a bridge between TJ proteins and cytoskeletal proteins [21, 22] . In addition, the interaction of claudins with ZO-1 or ZO-2 is indispensable for TJ protein formation [23] . Destruction of these proteins is closely related to ARDS [24, 25] . Damage to TJ proteins not only J o u r n a l P r e -p r o o f results in the formation of alveolar exudate but also attenuates its clearance [26, 27] .

The prognoses of patients with ARDS with low alveolar fluid clearance rates are significantly worse than of ARDS patients with higher alveolar fluid clearance rates [28] . As the protective effect of glycocalyx can relieve ARDS development, whether intact glycocalyx protects TJs has not been well studied. We hypothesize that the shedding of glycocalyx can lead to TJ damage. To confirm this assumption, animal and cell experiments were performed in this study.

Male C57BL/6 mice (18-23 g; 8-10 weeks old) were purchased from Jinan Pengyue Laboratory Animal Breeding Co., Ltd. (Shangdong, China) and were housed under standard conditions (22 ± 2°C; 50% ± 10% relative humidity; 12-h:12-h light/dark cycle). The mice were adapted to the environment for 2-3 days before the experiments. All animal experiments and feeding methods complied with the guidelines for the Care and Use of Laboratory Animals established by the US National Institutes of Health and were approved by the Binzhou Medical University Institutional Review Board.

Animals were randomly divided into six groups: control, lipopolysaccharide (LPS), heparinase III, LPS+NAH, heat-inactivated (HI) heparinase-III, and NAH groups. To J o u r n a l P r e -p r o o f induce LPS-mediated lung injury, the animals were intratracheally administered 5 mg/kg LPS (from Escherichia coli O55:B5, L2880, Sigma-Aldrich St. Louis, MO, USA) or saline (50 μL) and sacrificed 6 h later. The heparinase III group and the HI heparinase-III group were intratracheally administered 0.5 U heparinase III or 0.5 U HI heparinase-III (from Flavobacterium heparinum, Sigma-Aldrich, H8891), respectively, and sacrificed 1 h later [17] . We inactivated heparinase-III by heating it at 100 °C for 5 min. The LPS+NAH group and NAH group were pretreated with 150 µg NAH (Sigma-Aldrich, A8036, administered via 200 μL subcutaneous injection) [18, 29] , and one hour later, mice in the LPS and LPS+NAH groups were treated with 100 μg/mL LPS for 6 h. The bronchoalveolar lavage fluid (BALF), serum samples, and lung tissue samples were harvested.

Mouse lung tissues were collected and fixed with 4% paraformaldehyde for 48 h.

The tissues were processed, embedded in paraffin, and sectioned into 4-µm-thick slices. After hematoxylin and eosin staining, the slides were observed under an optical microscope. Lung injury was characterized by alveolar congestion and hemorrhage, alveolar neutrophil infiltration and aggregation, and thickening of the alveolar walls.

Samples were scored based on the presence or absence of each feature (0, minimal; 1, mild; 2, moderate; 3, severe; and 4, maximal). For each sample, six high-magnification fields were randomly scored for each feature, and the scores were combined to generate average lung injury scores [30] .

The lung tissues were harvested and weighed with filter paper; this weight was recorded as the wet weight (W). The tissues were dehydrated at 60°C for 48 h and weighed to obtain the dry weight (D). The W/D ratio was used to evaluate the degree of pulmonary edema.

After the mice were harvested, the alveoli were repeatedly lavaged with 500 μL PBS three times to obtain the BALF. The BALF of each sample was centrifuged at 900 ×g for 10 min at 4°C, and the supernatant was collected. The blood samples were collected from the eyeballs of the mice and centrifuged at 1500 ×g for 20 min.

The levels of HS in the BALF and serum were evaluated with corresponding ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Shanghai, China; ml111013) in accordance with the manufacturer's instructions.

Human pulmonary epithelial cells (A549 cells) were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Science (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a humidified incubator with 5% CO2 at 37°C. The cells were also separated into six groups: control, LPS, heparinase III, LPS+NAH, HI heparinase-III, and NAH group. The heparinase III and NAH concentrations used were based on the results of preliminary experiments and previous reports [18, 31] .

The control group was cultured in complete culture medium without LPS treatment, and cells in the LPS+NAH group and NAH group were pretreated with 10 μg/mL NAH for 1 h, after which the cells were thoroughly washed with PBS twice and replaced with complete medium. At 1 h, cells in the LPS and LPS+NAH groups were treated with 100 μg/mL LPS for 6 h. Cells in the heparinase III and HI heparinase-III groups were treated with 0.1 U/mL heparinase III or HI heparinase-III respectively for 1 h. In the recombinant HPA experiment, alveolar epithelial cells were divided into a control group and recombinant HPA group. Cells in the recombinant HPA group were treated with recombinant HPA (2 μg/mL, APA711Hu01, Cloud-clone Corp., Wuhan, China) for 12 h.

The supernatant of each group was collected, and heparanase concentration in the supernatant was measured with an human HPA Enzyme Assay kit (cat. no. E01H0100; BlueGene, Shanghai, China) according to the manufacturer's protocol. HS levels in the supernatant were evaluated with the corresponding ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd. ml061639) in accordance with the manufacturer's instructions.

Lung tissues were sectioned into 4-μm-thick slices, deparaffinized with xylene, and then dehydrated with an ethanol gradient. After thermal repair of the antigens, the samples were blocked with goat serum for 30 min at room temperature and than Bands were visualized using an electrochemiluminescence kit (Millipore, Billerica, MA, USA). Densitometry analysis was performed in the Image-J software.

To visualize epithelial glycocalyx and TJ protein structures between pulmonary epithelial cells via electron microscopy, the mice were anesthetized and perfused with a solution composed of 2% glutaraldehyde, 0.1 M sodium cacodylate buffer (pH 7.3), 2% sucrose, and 2% anthanum nitrate through a cannula placed in the left ventricle.

The lung tissue was fixed and cut into 1-mm 3 -sized slices. The slices were it were immersed in fixation solution for 2 h, soaked overnight in solution without glutaraldehyde, and washed with alkaline (0.03 mol/L NaOH) saccharose (2%) solution. After contrast enhancement with a solution containing 2% osmium tetroxide and 2% lanthanum nitrate, embedding in araldite, and microtomic sectioning, electron J o u r n a l P r e -p r o o f microscopy was performed.

For quantitative PCR analyses, total RNA was extracted from A549 cells using 

Statistical analysis was conducted with SPSS 22.0 software (SPSS Inc., Chicago, IL, USA). For normally distributed data, continuous variables were presented as mean ± standard deviation. One-way analysis of variance and least significant difference test were applied for multi-group comparisons. For data with skewed distributions, variables were expressed as median (25th and 75th percentiles). The Kruskal-Wallis test was applied for multi-group comparisons, and Mann-Whitney U test was performed for two-group. P ＜0.05 was considered statistically significant.

Inflammatory cell infiltration and alveolar exudation were increased and the alveolar septum was thickened in the LPS and heparinase III groups compared to the control group. These pathological changes were alleviated by NAH pretreatment (Fig.   1A -C). The W/D ratios of lung tissues from the LPS and heparinase III groups were significantly higher than in the control group, indicating increased pulmonary edema, whereas the ratios for mice pretreated with NAH were significantly lower than those of the LPS group (P＜0.05) (Fig. 1D ).

The HS content in BALF of mice confirmed that epithelial HS degradation increased after LPS stimulation and after heparinase III treatment compared to the control group. After using NAH, the epithelial HS content in BALF was lower than that in the LPS group (P＜0.05) (Fig. 2B) . Moreover, the ratio of HS in the BALF/serum (Fig. 2C ) and content of HS in the cell supernatant ( Fig. 2A) showed a consistent trend. However, there was no significant difference in the blood HS levels in each group (Fig. 2D) .

HS expression was significantly decreased in lung tissues from the LPS and heparinase III groups compared to the control group. Compared with the LPS group, the LPS+NAH group displayed attenuated reductions expression in HS (P＜0.05) (Fig.   3A, B) . Consistently, in A549 cells, HS expression was decreased in the LPS and J o u r n a l P r e -p r o o f heparinase III groups compared to the control group. This decrease in HS expression was inhibited by pretreatment with NAH (P＜0.05) (Fig. 3C, D) .

Immunofluorescence analysis revealed that in mice, occludin (Fig. 4A, B) , claudin 4 (Fig. 4C, D) and ZO-1 (Fig. 4E, F) Furthermore, we detect occludin (Fig. 5C, D) , claudin 4 (Fig. 5E , F) and ZO-1 (Fig.   5G , H) expression was confirmed in human alveolar epithelial cells through western blotting analysis. TJ proteins were significantly decreased after the loss of HS in the LPS and heparinase III groups, whereas the LPS+NAH group displayed increased expression of these proteins compared to the LPS group (P＜0.05).

The control group mice displayed a continuous, dense glycocalyx covering the alveolar epithelial cells. The glycocalyx of mice in the LPS group was completely or partially lost. Additionally, the glycocalyx was moderately affected in the NAH pretreatment group (Fig. 6A ).

The control group mice showed normal and intact TJ protein structures, whereas the LPS group showed loss of TJ structures, as evidenced by an intermittently widened distance between cells, indicating the loss in TJ protein integrity. However, these changes were improved in the NAH pretreatment group (Fig. 6B) .

HPA expression in the supernatants of alveolar epithelial cells in the control group and LPS group was detected by ELISA. Compared with the control group, HPA expression in the LPS group was significantly increased (P＜0.05) (Fig. 7) .

Through immunofluorescence analysis, we demonstrated that HS level in alveolar epithelial cells of the recombinant HPA group was significantly reduced compared to the control group (P＜0.05) (Fig. 8A, C) .

Immunofluorescence analysis showed that in the control group, the staining intensity of ZO-1 was strongly positive in human alveolar epithelial cells, with a continuous and undulating distribution along the cell membrane (Fig. 8B) . However, in the recombinant HPA group, ZO-1 lost its normal continuous distribution in epithelial cells.

Furthermore, we validated the expression of ZO-1 in human alveolar epithelial cells via western blotting (Fig. 8D, E) . The recombinant HPA group inhibited the protein expression of ZO-1, resulting in a particularly dramatic decrease in ZO-1 expression (P＜0.05).

The mRNA levels of ZO-1 in human alveolar epithelial cells were remarkably decreased after recombinant HPA challenge compared to the control group (***P＜ 0.001) (Fig. 8F) .

In this study, an animal ARDS model induced by intratracheal instillation of LPS [33] and A549 cell injury model were used to observe the LPS-induced glycocalyx and TJ changes in ARDS epithelial cells. The results showed that LPS can lead to increased degradation of the epithelium glycocalyx component HS and destruction of TJ proteins (ZO-1, occludin, claudin 4), increasing the permeability of the alveolar barrier and exudation of inflammatory cells.

alveolar epithelial cell research [34, 35] . We further observed the shedding of the cleavage product HS of the glycocalyx and increased the expression of HS in the cell supernatant after LPS stimulation. Through in vivo experiments, we found that HS expression in the BALF of the model group was significantly increased; however, the BALF/serum ratio was increased, but blood HS was not significantly increased, confirming that HS shedding after LPS stimulation occurred mainly because of the glycocalyx of the alveolar epithelium.

The barrier of the alveolar epithelium is composed of the alveolar epithelium, alveolar epithelial glycocalyx, and TJs of epithelial cells. HS is the main component of glycocalyx, which plays an important role in the epithelial barrier [36] . Studies J o u r n a l P r e -p r o o f have shown that increased HS damage can lead to increased alveolar permeability and ARDS formation [36, 37] , affecting the recovery of lung injury [38] ; these results are similar to those reported above in this study.

HPA is the only enzyme selectively shedding HS. In this study, the increase in the HPA content in the supernatant of cells indicates that HS destruction is related to the increase in HPA content stimulated by LPS. To confirm the role of HPA in LPS-induced lung injury, we added exogenous heparinase III to obtain HS degradation results similar to that of LPS. Further addition of the HPA competitive antagonist NAH [17] can reverse this phenomenon. Therefore, LPS can increase HPA, which in turn leads to HS degradation. The integrity of the glycocalyx of alveolar epithelial cells is destroyed, leading to increased alveolar barrier permeability followed by lung injury or ARDS. ZO-1 is the cytoskeleton protein of the cell junction protein, and its destruction can affect the binding of occludin and claudin 4 to ZO-1, resulting in formation of cell cracks [21, 22] . When ZO-1 was depleted in epithelial cells, barrier-forming proteins such as claudin and occludin could not assemble into the strands normally found in TJ proteins, and the epithelial barrier effect of TJ proteins was completely eliminated [23, 39] . To explore the mechanism of HPA-HS on ZO-1, we assessed the ZO-1 mRNA.

Our study indicated that HPA-HS may interfere with ZO-1 synthesis and then affect TJ proteins assembling and function. Syndecan 1 (SDC-1), another component of glycocalyx, acts in synergy with TJ through Stat3 signaling [40] . To explore whether HPA-HS uses the same signaling pathway as SDC-1 does for affecting TJs, we studied the HPA-HS-Stat3 pathway by performing in vitro experiments. The western blotting results showed that the decrease in the phosphorylation level of Stat3 in cells stimulated by recombinant HPA did not significantly differ from that of the normal group (data not shown). Therefore, the signaling pathway via which HPA affects ZO-1 differs from that of SDC-1. To identify the pathway through by which HPA-HS affects ZO-1 expression, further studies are needed.

This study has some limitations. First, we only studied changes in epithelial TJ proteins after alveolar epithelial glycocalyx HS shedding. Further analyses are required to determine whether other components of the glycocalyx, such as SDC-1, SDC-4, and chondroitin sulfate, are related to epithelial TJ proteins. Second, many proteins are involved in TJs; however, few have been studied. Therefore, whether glycocalyx also affects should be further analyzed.

Our results demonstrate that shedding of the alveolar epithelial glycocalyx affected the expression of TJ proteins (occludin, ZO-1, and claudin 4) and disrupted TJ, and this effect was reversed by NAH. The mechanism by which glycocalyx affects TJ proteins partly involves HPA, which suppresses the expression of ZO-1.

Protecting glycocalyx may reduce TJ damage and improve the alveolar barrier, reduce pulmonary edema, and provide new therapeutic targets for ARDS treatment.

None. µm. Data represent the mean ± SD of three independent experiments. # P < 0.05 compared to the control group; * P < 0.05 compared to the LPS group. 

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Immunofluorescence images of HS (A, green) and associated fluorescence intensity analysis of HS(C) in A549 cells in the control and recombinant HPA groups

Immunofluorescence images of ZO-1 (B, red) in human alveolar epithelial cells in the control and recombinant HPA groups

This work was supported by funding from the National Natural Science Foundation of China (no.81670078) and Taishan Scholar Project of Shandong Province.