TNF-α and RPLP0 driving apoptosis of endothelial cells as the shared molecular mechanism of high altitude pulmonary edema in sojourners and natives: bioinformatic analysis and experimental validation

doi:10.21203/rs.3.rs-4478935/v1

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TNF-α and RPLP0 driving apoptosis of endothelial cells as the shared molecular mechanism of high altitude pulmonary edema in sojourners and natives: bioinformatic analysis and experimental validation

https://doi.org/10.21203/rs.3.rs-4478935/v1

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published 07 Aug, 2024

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High altitude pulmonary edema (HAPE) is a fatal threat for those sojourners who ascend rapidly without sufficient acclimatization. High altitude natives are insusceptible to HAPE resulting from evolved genetic specializations of adaption. In this study, based on GSE52209, the gene expression profile of HAPE patients was compared with acclimatized sojourners and adapted natives, with the common and divergent differential expressed genes (DEGs) and their hub genes being identified, respectively. Bioinformatic methodologies of functional enrichment analysis, immune infiltration, diagnostic model constructing, ceRNAs and drugs predicting, were performed to detect the potential biological functions and molecular mechanisms. Next, an array of in-vivo experiments in HAPE rat model and in-vitro experiments in HUVECs were conducted to verify the results of bioinformatic analysis. Enriched pathways of DEGs and immune landscapes for HAPE exhibited significant differences between sojourners and natives, and the common DEGs were mainly enriched in the pathways of development and immunity. Nomograms found the upregulation of TNF-α and downregulation of RPLP0 exhibited high diagnostic efficiency for HAPE both in sojourners and natives, which was furtherly validated in the HAPE rat model. Treatment of TNF-α and knock-down of RPLP0 activated apoptosis signaling in endothelial cells (ECs) and enhanced endothelial permeability. Conclusively, TNF-α and RPLP0 are identified as the shared biomarkers and molecular basis for HAPE during the acclimatization/adaption/maladaptation processes in sojourners and natives, which inspire new ideas for the prediction and treatment of HAPE.

High altitude pulmonary edema

acclimatization

adaption

apoptosis

bioinformatic analysis

Thousands of sea level sojourners travel to altitude regions with elevation > 2500m for recreational, economic, military or religious purposes every year¹. Oxygen is vital for cellular metabolism, and as altitude increasing, the hypoxic stress caused by low inspired oxygen pressure would exert deleterious effects on all physiological functions. To defend against the oxygen deprivation at high altitude, a complex and finely integrated physiological process termed acclimatization would be mobilized in sea sojourners, which usually comprise the cardiopulmonary and hematological responses including hyperventilation, hemoconcentration, and an increase in heart rate and cardiac output². The hypoxic pulmonary vasoconstriction confers no advantage but may contribute to the occurrence and development of high altitude illnesses, hence it is not the feature of acclimatization but considered as a maladaptive response³.

High-altitude pulmonary edema (HAPE) is a potentially lethal manifestation of high-altitude illness which usually happens at any point 1–5 days following acute exposure to high altitude hypoxia, and is characterized by non-cardiogenic but high-permeability alveolar edema¹. Uneven hypoxic pulmonary vasocontriction and capillary leakage with a large concentration of high-molecular-weight proteins are two critical processes of the HAPE pathogenesis⁴. It is believed that those extremely thin blood-gas barriers are not protected from the uneven vasoconstriction-induced high capillary pressure and develop ultrastructural and mechanical disruptions³. Therefore, the endothelial dysfunctions in HAPE not only play a part in causing the excessive pulmonary vasocontriction through impaired release of relaxing factors and vasoconstrictors, but also exacerbate the vascular leakage through damaging pulmonary capillary permeability⁵.

There are over 150 million people around the world inhabited at the altitude > 3000 m for hundreds of generations⁶. Natives from the high altitude regions have developed many distinctive patterns and biological characteristics termed adaption in the response to hypoxia, which include augmented aerobic performance, larger lung volume and capacity, and enhanced hypoxic resistance with higher redox status and nitric oxide metabolites^{7, 8}. Besides, natives show attenuation of the maladaptive responses to hypoxia, including a smaller degree of hypoxic pulmonary vasoconstriction and reduced hemoglobin^{3, 8, 9}. Acclimatization of sojourners is a reversible physiological phenomenon, while adaptation of natives involves changes that take place over generations of natural selection^{7, 9}. Thus, the distinctive physiological plasticity between native and sojourners might be interpreted by the genetic variations of protein activity, organelle function, cellular metabolism, and the function of organ systems^{7, 10}. In recent years, evidences for the adaptation genetic basis and the comparing of genetic profiles between high altitude natives and lowlanders emerge^{7, 8}, in which EPAS1, EGLN1 and PPARA were identified as the strong candidate genes for the natural selection at high altitude³. There is much interest in whether genetic factors and which molecular signaling play a role in the pathogenesis of HAPE, but no definitive gene differences and specific biomarkers for HAPE have been reported so far^{3, 11, 12}.

Separating the molecular mechanisms of adaption from acclimatization processes may be beneficial for gaining a better understanding of offsetting hypoxic stress. The rapid development of sequencing technologies informs new approaches for exploring the pathophysiological processes of HAPE. Thus, in the present study, we selected the GSE52209 dataset to explore the blood expression profiling in the individuals who developed HAPE, as compared with the sea level sojourners who were acclimatized to high altitude and the adapted high altitude natives from Ladakh, respectively. Then, Venn analysis revealed the common and disparate DEGs in the pathogenesis of HAPE between acclimatized sojourners and adapted natives, and a set of bioinformatic methodology and experimental validation were applied to identify the hub genes and explore their potential biological functions and molecular mechanisms. Conclusively, through separating the transcript features of adaption and acclimatization, our study provides a novel perspective for understanding the HAPE pathogenesis, which may inspire new ideas for the prediction and treatment.

Datasets

The GSE52209 dataset, generated on the GPL9365 platform, was obtained from Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) and integral to our analysis. This dataset contains blood samples of 14 sea level sojourners who were acclimatized to high altitude and grouped as controls, 14 high altitude natives from Ladakh (HAN) and 17 individuals who developed high altitude pulmonary edema within 48–72 hours after air induction to high altitude (HAPE).

Differentially expressed genes (DEGs) analysis

Microarray data from the GEO database were retrieved and processed using the “GEOquery” package (version 2.66.0). DEG analysis was conducted using the “limma” package (version 3.54.0), according to the criteria of adjusted p-value < 0.05 and abs[log2FC] > 1. The “ggplot2” (version 3.4.0) and “ComplexHeatmap” (version 2.14.0) packages were used for the visualization and generation of volcano plots and heatmaps, respectively.

Functional enrichment analysis

To explore the biological significance of the DEGs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the “clusterProfiler” package (version 4.2.2). The p-value < 0.05 was considered significant.

Immune infiltration analysis

xCell is a novel and validated gene signature-based strategy used to infer immune and stromal cell types¹³. The infiltration levels of various immune cell types in HAPE group were evaluated using xCell as compared with control group and HAN group in GSE52209, respectively. Group boxplots were generated and compared using the Wilcoxon rank-sum test to determine significant differences between cell types, with a cutoff p-value value < 0.05.

Construction of protein–protein interaction (PPI) network and selection of hub genes

The protein–protein interaction (PPI) pairs of DEGs in the A/B/C gene sets were investigated online using STRING database (http://string-db.org) and the PPI networks were visualized using cytoscape (version3.6.1). Then, five algorithms including Maximal Clique Centrality (MCC), Maximum Neighborhood Component (MNC), Edge Percolated Component (EPC), Degree and Bottleneck, were used to calculate each gene. The TOP 10 genes in each algorithm were furtherly overlapped by Venn analysis and identified as the hub genes.

Nomogram construction and receiver operating characteristic (ROC) curve evaluation

To predict the probability of HAPE from the hub genes expression, nomogram models were constructed using the “rms” package. Calibration curve was plotted to assess the predictive ability of nomograms, in which the Hosmer-Lemeshow (HL) test was used to determine the dispersion degree between the predicted value and true value (p-value > 0.05 suggested that there is no difference between the predicted value and true value, and the model has excellent goodness-of-fit). Besides, to assess the diagnostic capability of the nomograms, ROC curves were constructed using “pROC” package, and area under curve (AUC) was calculated with values closer to 1 suggesting higher diagnostic accuracy.

Construction of ceRNA network

The elmmo database was used to predict mRNA-miRNA interaction pairs based on the hub genes. Then based on the TOP 5 miRNAs with highest binding score, the starBase database was used to screen the miRNA-lncRNA interaction and finally construct the ceRNA network of mRNA-miRNA-lncRNA. Cytoscape was used to visualize the ceRNA network.

Drugs and compounds prediction

The Drug Signatures Database (DSigDB, http://dsigdb.tanlab.org/DSigDBv1.0/ ) was used to predict the drugs which potentially act on the hub genes according to the criteria of adjusted p-value < 0.01. The Encyclopedia of Traditional Chinese Medicine database (ETCM, http://www.nrc.ac.cn:9090/ETCM/ ) was used to predict the traditional Chinese medicines (TCMs) which potentially act on the hub genes. Then, according to the threshold of bioavailability ≥ 30%, drug-likeness ≥ 0.18, the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP, https://www.tcmsp-e.com/ ) was used to screen the active ingredients and effective components of those predicted TCMs. Cytoscape was used to visualize the networks of Drugs-Genes and TCMs-ingredients-Genes.

HAPE rat model

Adult male SD rats (10 − 12 weeks old, body weight > 300 g) were used in this study. All rats were raised in specific pathogen-free (SPF) cages with suitable temperature (25 ± 5°C) and humidity (50 ± 5%). All experiments involving animals were approved by the Ethics Committee for Animal Care and Use of Air Force Medical University.

The HAPE rat model was established based on a previously described method¹⁴. But in the present study, we explored that the 3 days is the most appropriate timing for acute modelling which caused the most severe lung injury. Rats were placed in the hypobaric chamber system, which is developed by our laboratory and consists of a vacuum pump, a control panel, a pressure and flow control system and hypobaric chambers, to mimic the high-altitude environment¹⁵. The altitude was set to 6000 m with a height rising rate of 20 m/s, and the temperature and humidity were maintained at 25°C and 55%.

Wet/dry weight ratio of lung tissues

Following the euthanasia of rats, wet weight was measured immediately after excising the middle lobe of the right lung. Thereafter, the lung tissues were dried at 60 ℃ in a constant temperature dry oven for 48 h and weighed again as dry weight. Finally, the wet/dry (W/D) weight ratio of lung tissues was calculated.

Hematoxylin − eosin (HE) and immunofluorescence staining

After collecting the middle lobe of the right lung, the lung tissues were fixed with 4% paraformaldehyde for 24 h. After the paraffin sections were prepared, dewaxing, hematoxylin staining, eosin staining, and dehydration were sequentially performed. Images were captured using photomicroscopy (Olympus, Tokyo, Japan).

Rat lung tissues in paraffin-embedded sections were deparaffinized, rehydrated, fixed, and blocked with 5% BSA, and then incubated with primary antibodies against cleaved caspase-3 at 1:200 dilution (#9661, cell signaling technology, USA), RPLP0 at 1:200 dilution (11290-2-AP, proteintech, China) and CD31 at 1:500 dilution (28083-1-AP, proteintech, China) at 4°C overnight, followed by incubation with fluorescently labeled secondary antibodies. Then, 1 µg/mL Hoechst 33258 solution (Thermo Fisher Scientific, MA, USA) was used for nuclear staining. The stained sections were examined by Pannoramic MIDI and evaluated through Pannoramic Viewer (3DHISTECH, Budapest, Hungary).

Transmission electron microscopy (TEM)

A part of the inferior lobe tissue of the right lung was removed and immediately fixed with 4% glutaraldehyde (Servicebio, Wuhan, China) and then trimmed into 0.1 × 0.1 × 0.1 cm³ blocks for further fixation with 1% osmium tetroxide in deionized water. Subsequent steps were performed as previously described¹⁵. Electron micrographs were collected using TEM (HITACHI 7800, Tokyo, Japan) at 80 kV. The ultrastructure of lung tissues and pulmonary endothelium was observed at ×8k and ×25k magnifications.

Cell culture and lentiviral transduction

Human umbilical vein endothelial cells (HUVECs) were obtained from ScienCell (Carlsbad, CA, USA) and cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (FBS) (Thermo Scientific, Rockford, IL, USA), 100 U/mL penicillin (Solarbio, Beijing, China), and 100 µg/mL streptomycin (Solarbio, Beijing, China).

HUVECs were transduced with a lentivirus carrying RPLP0 shRNA. Cells with the stable knock-down of RPLP0 were screened with puromycin resistance and furtherly confirmed by western blotting analysis.

Detection of endothelial permeability in vivo ¹⁶ and in vitro¹⁷

The rats were injected with Evans blue dye (20 mg/mL) through the femoral vein (60 mg/kg). One hour after injection, the vasculature was perfused with saline to remove intravascular Evans blue dye, and upper lobe tissue of the right lung was collected and homogenized. The protein concentration of lung tissue homogenate was assessed by BCA assay. The concentration of Evans blue dye in the lung tissue supernatant was determined by measuring the absorbance at 620 nm and normalized to the protein concentration.

8×10⁴ HUVEC cells were seeded into 12-well transwell filters (3-µm pore size; Corning) and then incubated when cell confluency reached 100%. Evans blue dye-labeled albumin (0.67 mg/mL) was added into the upper chamber, and after 1 h of incubation, the lower medium was collected and the concentration of Evans blue dye was determined by measuring the absorbance at 620 nm and normalized to a standard curve of known Evans blue dye concentrations.

TUNEL staining

The apoptosis of HUVECs and ECs in rat lung tissues were detect by TUNEL staining according to the manufacturer’s instructions using a commercially available kit. The numbers of TUNEL-positive ECs and total cells were counted, and apoptosis was evaluated by the ratio of positive cells to total cells.

ELISA assay

Protein level of TNF-α in the rat serum and lung tissues were measured with ELISA kits according to the manufacturer’s instructions.

Western blotting and RT-qPCR analysis

HUVECs and rat lung tissues were dissociated by lysis reagent, and then protein was extracted and quantified using a BCA kit (Thermo Fisher Scientific, MA, USA). Equivalent amounts of proteins from different groups were loaded, electrophoresed and transferred to a polyvinylidene difluoride membrane (Millipore, Schwalbach, Germany). The membrane was blocked with 5% BSA in Tris-buffered saline with 0.1% Tween 20, followed by incubation with primary antibodies against cleaved caspase-3 at 1:2000 dilution (#9661, cell signaling technology, USA) and RPLP0 at 1:1000 dilution (11290-2-AP, proteintech, China) overnight at 4°C. Then, the membrane was incubated with appropriate HRP-linked secondary antibodies for 1.5 h at room temperature. The membrane was finally imaged using an Odyssey scanner (LI-COR Biosciences, NE, USA) and analyzed using NIH ImageJ softwar.

HUVECs and rat lung tissues were homogenized with RNAiso (Takara, Otsu, Japan).

Complementary DNA (cDNA) was amplificated with SYBR Premix Ex TaqTM (TaKaRa Bio, Otsu, Japan). RT-qPCR was performed using SYBR Green PCR master mix (Life Technologies). Actb was used as the control for PCR product quantification and normalization. Data were analyzed via the relative Ct (2 − ΔΔCt) method and were expressed as a fold change compared with the respective control.

Statistical Analysis

All quantitative data are presented as the mean ± SEM. Comparisons between 2 groups were performed using 2-tailed unpaired Student’s t tests. Statistical differences among groups were analyzed by 1-way ANOVA followed by Bonferroni’s post hoc test. All statistical analyses were performed with Prism software (GraphPad Prism for Windows, version 9.0, Nashville, TN). Differences were considered significant at *P < 0.05, **P < 0.01, and ***P < 0.001¹⁸.

1. Differential gene expression profiles and immune landscapes for HAPE between acclimated sojourners and adapted natives The expression profiling of blood samples in sea level sojourners who were acclimatized to high altitude (n = 14), high altitude natives (n = 14) and individuals who developed high altitude pulmonary edema (n = 17) were published in the GSE52209 dataset, and furtherly analyzed by bioinformatic methodology and experimental validation in our present study (Fig. 1). First, based on the criteria of P-adjustment < 0.05 and abs [log2 FC] > 1, we screened 270 differential expresses genes (DEGs) between HAPE and acclimated sojourners (control) groups, and 166 DEGs between HAPE and high altitude natives (HAN) groups. Volcano plots and heat maps of these DEGs were shown, along with the top 20 upregulated and downregulated genes being highlighted in Fig. 2A-2D. Functional enrichment analysis of these DEGs indicated that between HAPE patients and acclimated sojourners, the DEGs were mainly enriched in the GO terms of axon development, transmembrane transporter activity and channel activity, and in the KEGG terms of neurodegeneration and Alzheimer disease (Fig. 3A); while between HAPE patients and adapted natives, the DEGs mainly were enriched in the GO terms of cytokine-mediated signaling pathway and embryonic organ development, and in the KEGG terms of TGF-beta signaling pathway (Fig. 3B). Next, xCell algorithm was used to analyze the infiltration of 64 immune cell types, and results indicated that as compared with acclimatized sojourners, the abundance of CD4⁺ native T cells, CD4⁺ T cells, macrophages M2, megakaryocytes, NKT, pro B cells, skeletal muscle cells, smooth muscle cells and Tregs were significantly increased in HAPE patients (Fig. 4A and 4B); while as compared with adapted natives, only three types of immune cells including CD4⁺ native T cells, CD4⁺ T cells and eosinophils were significantly increased in HAPE patients (Fig. 4C and 4D). And among these HAPE-related immune cells, Venn diagram showed there were 4 common immune cell types, including CD4⁺ native T cells, CD4⁺ T cells, MEP and Th2 cells, altering unanimously in the sojourners and natives (Fig. 4E). These results suggested the expression profile and immune landscape for HAPE were different between sojourners and natives.

2. Screening and functional analysis of common/divergent DEGs for HAPE between acclimated sojourners and adapted natives.

Sea level sojourners and high altitude natives display distinctive characteristics of physiological responses to hypoxia⁸. In order to separate the transcript features for HAPE in sojourners and natives, the DEGs between HAPE and control groups, and between HAPE and HAN groups were subjected to Venn analysis with opposite expression trends being excluded (Fig. 5A). A total of 108 common DEGs were identified to be obviously altered in HAPE group as compared with both control and HAN groups, while 162 DEGs merely altered between HAPE and control group, and 58 DEGs merely altered between HAPE and HAN group (Fig. 5B). For further analyses, those common/divergent DEGs were classified and marked as gene sets of A (control-specific), B (HAN-specific) and C (common), respectively (Fig. 5B). Functional enrichment analysis of these common/disparate DEGs indicated that the control-specific DEGs in A gene set were mainly enriched in the pathways of channel activity, axonogenesis, and neurodegeneration disease (Fig. 5C); the HAN-specific DEGs in B gene set were mainly enriched in the cytokine-mediated signaling pathway and adherens junction (Fig. 5D); the common DEGs in C gene set were mainly enriched in embryonic organ development, leukocyte migration and TGF-beta signaling pathway (Fig. 5E). TGF-ß is a multifunctional cytokine which is particularly critical to embryonic development, wound healing, tissue homeostasis, and immune homeostasis¹⁹. Thus, these results suggested that the signaling pathways of development and immunity were closely associated with the HAPE pathogenesis in both sojourners and natives.

3. Identification of hub genes in common/divergent DEGs.

PPI networks of these common/divergent DEGs in A/B/C gene sets were constructed using STRING and Cytoscape, respectively (Fig. 6A, 6C and 6E). Next, based on the five CytoHubba models: MCC, MNC, Degree, EPC and BottleNeck, we ranked the top 10 genes within the whole network and performed Venn analysis to obtain the overlapped genes (Fig. 6B, 6D and 6F). Finally, through the topological analysis algorithms, four genes of POLR1A, YMEIL1, JARIDIA and ESPL1 in A gene set, five genes of SNAI1, TCHP, ACAT2, ILK and DBI in B gene set, and four genes of TNF, RPLP0, ALDH3A1 and YBX1 in C gene set were identified as the hub genes for HAPE in the sojourners and natives, respectively (Fig. 6B, 6D and 6F).

4. TNFα-upregulation and RPLP0-downregulation exhibited significant diagnostic efficacy for HAPE in both sojourners and natives.

Based on the expression of aforementioned hub genes in A/B/C gene sets, nomograms for HAPE in sojourners and natives were constructed, respectively (Fig. 7A, 7D, 7G and 7J). In these nomograms, the score of each sample was calculated with a higher score indicating a higher likelihood of HAPE. Calibration curves were used to investigate the predicted probability of the nomogram models, and the slope close to 1 and CIC converge with trend of real situation suggested excellent predictive efficacy of these models (Fig. 7B, 7E, 7H and 7K). Besides, ROC curves were used to evaluate the diagnostic sensitivity and specificity of the nomogram models, and the AUC values were as follows: 0.983, 0.895, 1 and 1, all suggesting a superior diagnostic efficacy for identifying HAPE patients from sojourners and natives (Fig. 7C, 7F, 7I and 7L).

In the nomograms, the line segment length represents the contribution degree of each hub gene to the outcome event, and among the four hub genes in C gene set, we found TNF and RPLP0 showed high efficacy for the prediction and diagnosis of HAPE both in sojourners and natives (Fig. 7G and 7J). Besides, TNF expression was significantly increased while RPLP0 significantly decreased in the HAPE group as compared with both the acclimated sojourners and adapted natives (Fig. 8A), suggesting a potential role of TNF and RPLP0 as the common biomarkers for HAPE in sojourners and natives.

5. Validation of TNF-α-upregulation and RPLP0-downregulation in HAPE rats.

In order to establish HAPE animal model, Rats were subjected to the simulated altitude of 6000 m for 3 days by hypobaric chamber, with the lung wet to dry weight ratio and the lung histological changes being examined as previously reported¹⁴. As shown in Fig. 8B, after 3-day exposure to the simulated altitude of 6000m, the wet to dry weight ratio of rat lung tissues was significantly increased; Hematoxylin and eosin (HE) staining also showed obvious ultrastructure damages, including the alveolar edema and hemorrhage, and the lung interstitial thickening and deformation, occurred in the rat lung tissues (Fig. 8C), suggesting the rat model of HAPE was constructed successfully. Next, RT-qPCR analysis indicated the TNF-α mRNA expression significantly increased and RPLP0 expression significantly decreased in the lung tissues of HAPE rats (Fig. 8D and 8G). ELISA assay showed the protein level of TNF-α significantly increased in the lung tissues and serum of HAPE rats (Fig. 8E and 8F). Western blotting analysis showed the protein level of RPLP0 significantly decreased in the lung tissues of HAPE rats (Fig. 8H). These results were consistent with and hence verified the results of bioinformatic analysis in GSE52209.

6. Pulmonary ECs apoptosis and endothelial permeability were enhanced in the lung tissues of HAPE rats. HAPE is a high-permeability pulmonary oedema characterized by exaggerated vascular leakage, and it is reported that ECs apoptosis plays an important role in the regulation of endothelial permeability^20–22. In the present study, by transmission electron microscope (TEM), we observed obviously destroyed capillary endothelium and their tight junctions, as well as the apoptosis signs with nuclear chromatin condensation and fragmentation, perinuclear space broadening in the ECs of HAPE rat lung tissues (Fig. 9A). Next, TUNEL staining (Fig. 9B), along with the double-immunofluorescence staining and immunoblotting of apoptosis-related protein (cleaved caspase-3) (Fig. 9C and 9D) were conducted to assess the ECs apoptosis in the rat lung tissues, and results showed the ECs apoptosis rate and the expression of cleaved Caspase-3 in endothelium both were significantly increased in the HAPE rats. The lung endothelial permeability was examined by measuring the Evans blue dye accumulation and leakage levels in the lung tissues, and results suggested a higher endothelial permeability and vascular leakage in the lung tissues of HAPE rats (Fig. 9E). These results indicated the activation of pulmonary ECs apoptosis and the high endothelial permeability in the lung tissues of HAPE rats.

7. Treatment of TNF-α and knock-down of RPLP0 induced ECs apoptosis and increased endothelial permeability

TNF-α could directly activate apoptosis signaling²³. In the in-vitro experiments, the cultured HUVECs were treated with 40 ng/ml TNF-α for 24h, TUNEL staining and western blotting analysis showed the apoptosis rate and the expression of cleaved caspase-3 in HUVECs both were significantly increased by the stimulation of TNF-α (Fig. 10A and 10B). Next, HUVECs were seeded in the transwell chamber to form the endothelial monolayer in vitro. The endothelial permeability was assessed by measuring the Evans blue dye leakage levels from the upper chamber to the lower chamber, and results suggested that treatment of TNF-α significantly increased the endothelial permeability in vitro (Fig. 10C).

Double-immunofluorescence staining of RPLP0 and CD31 indicated the RPLP0 expression significantly decreased in the pulmonary endothelium of HAPE rats (Fig. 11A). Next, RPLP0 expression was silenced in HUVECs by lentivirus carrying RPLP0 specific shRNA. As examined by TUNEL staining and western blotting analysis, the HUVECs apoptosis rate and the cleaved caspase-3 expression both were significantly increased in the RPLP0 shRNA-HUVECs (Fig. 11B and 11C). Next, HUVECs infected with RPLP0 shRNA were seeded to establish the endothelial monolayer, and Evans blue dye leakage was measured using a transwell system. Results showed with the knock-down of RPLP0, the leakage of Evans blue dye through HUVECs monolayer was markedly increased, suggesting a higher endothelial permeability (Fig. 11D). Conclusively, there results indicated that TNF-α stimulation and RPLP0-downregulation both could activate ECs apoptosis signaling and thus enhance the endothelial permeability.

8. The prediction of competing endogenous RNA (ceRNA) and drugs targeting hub genes

Targeting the hub genes in A/B/C gene sets, we predicted mRNA-miRNA interaction pairs by elmmo database and selected the TOP 5 miRNAs with highest binding score to furtherly predict the miRNA-lncRNA interaction by starBase database. And finally, the ceRNA networks of mRNA-miRNA-lncRNA were constructed and visualized by Cytoscape (Fig. 12A-12C).

Using the DSigDB database, the potentially effective drugs targeting the hub genes were predicted (Fig. 13A-13C). Besides, using ETC and TCMSP databases, the candidate compounds of traditional Chinese medicines (TCMs) targeting hub genes also were predicted (Fig. 13D). And finally, the networks of Drugs-Genes and TCMs-Compounds-Genes were established and visualized by Cytoscape.

HAPE is a fatal threat for people ascending to the high altitude, which usually occurs in these sea level sojourners who ascend rapidly without sufficient acclimatization²⁴. In the present study, we used GSE52209 to compare the gene expression profiles of HAPE patients with acclimatized sojourners and adapted natives, respectively. High altitude natives and sea level sojourners exhibit distinct physiological traits in the response to hypoxia, which may result from the evolved genetic specializations of adaption and acclimatization⁸. Thus, the DEGs between HAPE patients and acclimatized sojourners may reveal the molecular basis of HAPE pathogenesis and the molecular mechanisms of acclimatization process in sojourners, and the DEGs between HAPE patients and adapted natives may reveal the molecular mechanisms of adaption process in natives. Here, we found the DEGs between HAPE and acclimatized sojourner groups were enriched in channel activity, while the DEGs between HAPE and adapted native groups were enriched in cytokine signaling. Immune infiltration analysis indicated the immune landscape for HAPE also were obviously different between sojourners and natives, and a total of 4 common immune cell types, including CD4⁺ native T cells, CD4⁺ T cells, MEP and Th2 cells, were identified to be involved in the both acclimatization and adaption processes.

Next, the two groups of DEGs were overlapped by Venn analysis to screen the common and divergent DEGs for HAPE between acclimatized sojourners and adapted natives, which may reveal the common and distinct molecular mechanism of the acclimatization and adaption processes. Functional analysis of these DEGs indicated the DEGs which merely altered between HAPE and acclimatized sojourner groups were enriched in channel activity, the DEGs merely altered between HAPE and adapted native groups were enriched in cytokine and adherens pathways, and the common DEGs overlapped between acclimatized sojourner and adapted native groups were enriched in the pathways of development and immunity¹⁹. These findings indicate that channel activity is critically implicated in the acclimatization process of sojourners, and the signaling pathways of development and immunity are implicated in the both acclimatization and adaption processes of sojourners and natives at high altitude. The long-term residence at high altitude for hundreds of generations provides sufficient time for genetic selections in high altitude natives, and enable them better surviving and performing in hypoxia⁷. A number of comparative studies have reported the different regulatory plasticity of physiological systems to hypoxia between high altitude natives and sea level sojourners^{7, 8}. The pathophysiology of HAPE is currently attributed to heterogeneous hypoxic pulmonary vasoconstriction and the following intercellular mechanisms of enhanced endothelial permeability, inflammation, and damaged fluid reabsorption capacity and matrix architecture²⁴. Channel activity plays a role in regulating the hypoxic pulmonary vasoconstriction²⁵, and cytokines promote lung endothelial permeability and leakage²⁶. The enrichment of channel activity in sojourners but not in natives is consistent with the physiological manifestations that excessive and uneven hypoxic pulmonary vasoconstriction trigger HAPE in sojourners, but high altitude natives show attenuated hypoxic pulmonary vasoconstriction and insusceptibility to HAPE^{8, 24}. The enrichment of development and immunity pathways in both sojourners and natives suggests that some phenotypic features, such as larger lung volume and capacity, abundant pulmonary capillaries and higher nitric oxide metabolites in natives, and the inflammatory signaling are critically implicated in the both acclimatization and adaption processes of sojourners and natives at high altitude.

Next, based on the topological analysis algorithms, hub genes were screened out in these common/divergent DEGs, and nomogram model indicated the upregulation of TNF-α and downregulation of RPLP0 exhibited high diagnostic efficiency for HAPE both in sojourners and natives. Tumor necrosis factor (TNF) plays a central role in orchestrating mammalian inflammatory responses. It promotes inflammation either directly by inducing inflammatory gene expression or indirectly by triggering cell death of apoptosis, necroptosis, and pyroptosis, which induces the production of proinflammatory mediators by activating innate immune receptors on neighboring cells²³. Furthermore, by causing the death of epithelial and endothelial cells, TNF also affect barrier integrity and additionally initiate inflammatory signaling through PRR-mediated sensing of pathogen-associated molecular patterns (PAMPs) from microbes ²³. It has been generally reported that hypoxia activated inflammatory signaling and the HAPE patients showed an obvious increase in inflammatory cytokine levels including TNF-α²⁶, which is consistent with our present finding. Ribosomal protein P0 (RPLP0) is a component of the ribosome 60S subunit located in the cytoplasm and nucleus, which appears to be involved not only in protein synthesis but also in transcriptional processes, DNA repair, cell development, cell apoptosis, and tumorigenesis^{27, 28}. High expression level of RPLP0 has been detected in many tumors²⁸, and the depletion of RPLP0 could lead to apoptosis and cell cycle arrest in many cancer cells^{27, 28}. Besides, there are evidences suggest that RPLP0 could be expressed on the surface of vascular endothelial cells under normal or specific conditions²⁹. In the present study, through an array of in-vivo and in-vitro experiments, we confirmed the upregulation of TNF-α and downregulation of RPLP0 in the lung tissues of HAPE rats, and furtherly found the addition of TNF-α and knock-down of RPLP0 could activate ECs apoptosis signaling and enhance endothelial permeability in the HAPE pathogenesis. Previous studies ever reported that B2M could act as a clinical potential biomarker for the early diagnosis and therapeutic evaluation of HAPE, and its decrease may be related to the reduced immunity due to reduced ability of MCH I in antigen submission¹². But this study did not describe which population, sojourners or natives, they drew their conclusions from. And in the highlanders of Andean, it is reported that the HAPE prevalence was strongly related to an increment of mean corpuscular haemoglobin concentration (MCHC)³⁰, but the molecular basis has not been studied. Our present findings not only confirm the diagnostic value of TNF-α and RPLP0 as the biomarkers for HAPE, but also clarify the shared molecular mechanisms that TNF-α-upregulation and RPLP0-downregulation driving ECs apoptosis to enhance endothelial permeability in the acclimatization and adaption processes of sojourners and natives at high altitude. For inspiring new treatment strategies, the networks of ceRNAs and drugs targeting these hub genes also were predicted and constructed in our present study.

However, some limitations of the study warrant further consideration. First, although we validated the hub genes expression in the HAPE rat model, further experiments using human samples are still required to confirm our findings due to the inherent limitations of bioinformatics techniques. Second, since our data were sourced from a public database, the covariates of age and comorbidities were not considered. Further clinical studies and higher levels of evidence are needed.

In summary, the differential gene expression profiles of HAPE patients between acclimatized sojourners and adapted natives indicated the divergent mechanisms of acclimatization and adaption in sojourners and natives at high altitude, and provided a novel perspective for understanding the pathogenesis of HAPE. The identification and validation of TNF-α and RPLP0 as the hub genes which show high diagnostic efficacy for HAPE in both sojourners and natives, and the following mechanisms exploration that the upregulation of TNF-α and downregulation of RPLP0 activated ECs apoptosis signaling and enhance endothelial permeability in the HAPE pathogenesis, both revealed the role of TNF-α and RPLP0 as the shared molecular mechanisms of HAPE during the acclimatization/adaption/maladaptation processes.

HAPE: high altitude pulmonary edema

ECs: endothelial cells

TNF-α: tumor necrosis factor-alpha

RPLP0: ribosomal protein lateral stalk subunit P0

DEGs: differential expressed genes

GO: Gene Ontology

KEGG: Kyoto Encyclopedia of Genes and Genomes

PPI: protein–protein interaction

ceRNA: competing endogenous RNA

Ethics approval and consent to participate

All animal experiments in this study were performed with the approval of ethical Committee of Experimental Animal Care of Air Force Medical University.

Availability of data and materials

The dataset GSE52209 was sourced from the GEO database https://www.ncbi.nlm.nih.gov/geo/.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported by grants from the National Natural Science Foundation of China (82072101) and the National Key Research and Development Program of China (2020YFA0803603 and 2021YFA1301402).

Author contributions

M-J.X., Y.L., Y-L.G. and P-J.L. designed and performed the experiments, prepared the ﬁgures, and wrote the manuscript. Y-R.B., B.Z., J.X., S-Y.H., Y-G.B., and Q-L.C. contributed to the performance of the experiments., J.M., and L.Z. supervised the work.

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TNF-α and RPLP0 driving apoptosis of endothelial cells as the shared molecular mechanism of high altitude pulmonary edema in sojourners and natives: bioinformatic analysis and experimental validation

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Journal Publication

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Abstract

Figures

Introduction

Materials and methods

Datasets

Differentially expressed genes (DEGs) analysis

Functional enrichment analysis

Immune infiltration analysis

Construction of protein–protein interaction (PPI) network and selection of hub genes

Nomogram construction and receiver operating characteristic (ROC) curve evaluation

Construction of ceRNA network

Drugs and compounds prediction

HAPE rat model

Wet/dry weight ratio of lung tissues

Hematoxylin − eosin (HE) and immunofluorescence staining

Transmission electron microscopy (TEM)

Cell culture and lentiviral transduction

TUNEL staining

ELISA assay

Western blotting and RT-qPCR analysis

Statistical Analysis

Results

7. Treatment of TNF-α and knock-down of RPLP0 induced ECs apoptosis and increased endothelial permeability

8. The prediction of competing endogenous RNA (ceRNA) and drugs targeting hub genes

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1