Purinergic P2X7 receptor mediates hyperoxia-induced injury in pulmonary microvascular endothelial cells via NLRP3-mediated pyroptotic pathway

Wen Zeng; Zhuyu Deng; Huaying Li; Shuqiang Gao; Rong Ju

doi:10.1515/med-2024-1097

Article Open Access

Purinergic P2X7 receptor mediates hyperoxia-induced injury in pulmonary microvascular endothelial cells via NLRP3-mediated pyroptotic pathway

Wen Zeng , Zhuyu Deng , Huaying Li , Shuqiang Gao and Rong Ju

Published/Copyright: December 4, 2024

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From the journal Open Medicine Volume 19 Issue 1

Abstract

Background

Hyperoxia-induced injury is a well-recognized cause of bronchopulmonary dysplasia (BPD). Existing research studies have not well elucidated the exact mechanisms underlying hyperoxia-induced cellular damage. This study examines the involvement of the P2X7 receptor (P2X7R) in hyperoxia-induced damage to human pulmonary microvascular endothelial cells (HPMVECs) via the NOD-like receptor family, pyrin domain-containing protein 3 (NLRP3) pathway.

Methods

HPMVECs developing hyperoxia-induced injury were subjected to the treatment of either selective inhibitors or a P2X7R/NLRP3 agonist. Western blot analysis assisted in the quantification of the levels of P2X7R, NLRP3, caspase-1, and gasdermin D (GSDMD). Additionally, the release of TNF-α, IL-1β, and IL-18 was assessed by ELISA and qRT-PCR.

Results

Exposure to hyperoxia diminished cell viability and escalated the levels of P2X7R, caspase-1, NLRP3, GSDMD, and N-terminal-GSDMD. This exposure notably increased the release of TNF-α, IL-1β, and IL-18 in HPMVECs. Notably, the suppression of P2X7R using the inhibitor A438079 decreased pyroptosis and inflammatory responses. Conversely, stimulation of P2X7R by 3′-O-(4-benzoylbenzoyl) adenosine 5′-triphosphate (BzATP) triggered pyroptosis, while inhibition of NLRP3 with glibenclamide ameliorated the damage induced by BzATP.

Conclusions

The P2X7R/NLRP3 pathway crucially affects the hyperoxia-induced inflammation and pyroptosis in HPMVECs, hinting the potential of blocking P2X7R/NLRP3-mediated pyroptotic pathway as a valuable therapeutic strategy for BPD.

Keywords: hyperoxia; P2X7 receptor; NLRP3 inflammasome; pyroptosis; pulmonary microvascular endothelial cell

1 Introduction

Bronchopulmonary dysplasia (BPD) is a class of chronic lung disease characterized by high morbidity and mortality among preterm infants. It has been reported that 30–50% of extremely premature infants with birth weights below 1,000 g develop BPD [1,2]. Research indicates that injury to pulmonary microvascular structures disrupts both lung function and postnatal lung development, including angiogenesis and alveolarization, which are critical pathological features of BPD [3–5]. Yet, the specific pathological mechanisms of BPD remain elusive, and effective treatments are still lacking.

Pulmonary microvascular endothelial cells (PMVECs) constitute a primary component of the alveolar-capillary membrane and are crucial for vascular homeostasis, angiogenesis, and cellular communication [6]. Dysfunction in PMVECs can contribute to BPD development. Hyperoxia is recognized as a significant etiological factor in BPD. Studies have demonstrated that hyperoxia diminishes the bioactivity of PMVECs and hinders the formation of distal microvasculature [7,8]. Despite significant advances, a full understanding of the molecular dynamics through which hyperoxia inflicts damage in PMVECs remains elusive.

Hyperoxia is linked to a surge in inflammatory cytokines, critical in the etiology of BPD [9,10,11]. The NLRP3 inflammasome, encompassing NLRP3, the adaptor ASC, and caspase-1, serves as a key regulator of pathological inflammation [12,13]. Activation of NLRP3 instigates the pro-caspase-1 to be converted to its active form (caspase-1), facilitating the generation of IL-1β and IL-18, and initiating GSDMD-driven pyroptosis [13,14].

The purinergic receptor P2X7R, an adenosine triphosphate (ATP)-gated ion channel, is integral to multiple cellular processes (immune response, proliferation, and apoptosis). It is posited that P2X7R may trigger the NLRP3 inflammasome, precipitating caspase-1-mediated pyroptosis [15–17]. Found in diverse microvascular endothelial cells, including PMVECs [18,19], the activation state of P2X7R under hyperoxic conditions remains uncertain.

This research developed a hyperoxic model in PMVECs to delineate the function of P2X7R in hyperoxic injury and to evaluate the interplay between P2X7R and the NLRP3 inflammasome in modulating PMVEC pyroptosis.

2 Materials and methods

2.1 Cell culture and experimental design

HPMVECs (cat. no. BNCC360121; BeNa Culture Collection, China) were cultured following the supplier’s instructions. All things considered, the HPMVECs were cultured in a humidified 5% CO₂ incubator at 37°C in endothelial cell medium (cat. no. 1001; ScienCell, San Diego, CA, USA) with 10% fetal bovine serum (cat. no. 25030081; Thermo Fisher Scientific, Waltham, MA, USA) and 100 U/mL penicillin/streptomycin (cat. no. C0222; Beyotime Institute of Biotechnology, Beijing, China). Cells were passaged every 2–3 days and used for passages 3–5.

There were five groups of HPMVECs randomly assigned to the study: control, hyperoxia, hyperoxia + A438079, BzATP, and BzATP + glibenclamide. Under normoxic circumstances (21% O₂ and 5% CO₂), the control, BzATP, and BzATP + glibenclamide groups were studied. Both the hyperoxia and hyperoxia + A438079 groups were exposed to a high level of oxygen (95% O₂ and 5% CO₂) for 24 and 48 h, respectively. Cells in the hyperoxia + A438079 group underwent 2 h of pre-incubation using 10 µM A438079 (cat. no. HY-15488; MedChemExpress, Monmouth Junction, NJ, USA) prior to hyperoxia; cells in the BzATP group received 200 µM BzATP (cat. no. HY-136254; MedChemExpress) for 2 h; and cells in the BzATP + glibenclamide group underwent 2 h of treatment using 200 µM BzATP and 50 µM glibenclamide (cat. no. HY-15206; MedChemExpress).

2.2 Immunocytochemistry (ICC)

To fix the HPMVECs, 4% paraformaldehyde was utilized, and to permeabilize them, 0.25% Triton X-100 was used. Cells received half an hour of inhibition using 1% bovine serum albumin (cat. no. 30063481; Thermo Fisher Scientific), followed by one night of pre-incubation using primary antibodies for CD34 (1:300; cat. no. sc-19621; Santa Cruz Biotechnology, Dallas, TX, USA) and Factor VIII (1:1,000; cat. no. ab275376; Abcam, Cambridge, MA, USA) at 4°C, and another 1 h of incubation using HRP-conjugated secondary antibodies (1:100, cat. no. A0208 and A0216; Beyotime Institute of Biotechnology). Diaminobenzidine (cat. no. sc-24982; Santa Cruz Biotechnology) was used to stain the cells for 5 min. Hematoxylin (cat. no. sc-24973; Santa Cruz Biotechnology) was then used as a counterstain. The photos were shot using an optical microscope (Leica, German).

2.3 Transmission electron microscopy (TEM)

Weibel-Palade bodies were detected by TEM. HPMVECs were subjected to 4 h of fixation in 2.5% glutaraldehyde in 0.1 M cacodylate buffer pH 7.4 at 4°C, followed by another 2 h of culture in medium added with 1% osmium tetroxide at 4°C. We used a graduated ethanol series of 30, 50, 70, 80, 95, and 100% to dehydrate the cells, and then embedded them in SPI-Pon^TM 812 epoxy resin (SPI Supplies, West Chester, PA, USA). Ultrathin sections of 70 nm thickness were prepared. Finally, a Hitachi HT7700 TEM (Japan) operating at 100 kV was used to observe certain ultrastructural features of sections stained using 3% uranyl acetate and 2.7% lead citrate.

2.4 Cell viability assay

The Cell Counting Kit-8 (CCK-8) assay (cat. no. CK04; Dojindo, Osaka, Japan) assisted in determining the cell viability as per the producer’s protocol. HPMVECs were seeded in 96-well plates (100 μL/well) to achieve 1 × 10⁴ cells per well, with each well added with 10 μL of CCK-8 reagents. Following 1 h of incubation, a microplate reader (Bio-Rad, Hercules, CA, USA) was adopted for measurement of the absorbance at 450 nm. The results were used to ascertain cell viability with a control value of 100%.

2.5 Quantitative real-time reverse transcription PCR (qRT-PCR) analysis

Following treatments, HPMVECs were treated with TRIzol Reagent (cat. no. 15596026; Thermo Fisher Scientific) for total RNA extraction, and cDNA was produced with the Takara Reverse Transcription Kit (cat. no. RR037A; Takara Bio, Kyoto, Japan). qRT-PCR was conducted using SYBR Green PCR Master Mix (cat. no. 1725150; Bio-Rad) (Table 1). Here we took GAPDH an internal control. Calculation of the relative expression relied on the 2^−ΔΔCq method.

Table 1

List of primers

Gene	Primer sequences	Product size (bp)
GAPDH	Forward: 5′-AGATCCCTCCAAAATCAAGTGG-3′	130
	Reversed: 5′-GGCAGAGATGATGACCCTTTT-3′	130
TNF-α	Forward: 5′-ACTGAAAGCATGATCCGGGA-3′	141
	Reversed: 5′-GCAGAAGAGCGTGGTGGC-3′	141
IL-1β	Forward: 5′-TCCGACCACCACTACAGCAAG-3′	91
	Reversed: 5′-GTGGGCAGGGAACCAGCATC-3′	91
IL-18	Forward: 5′-AGATAGCCAGCCTAGAGGTATGGC-3′	119
	Reversed: 5′-TGATGTTATCAGGAGGATTCATTTC-3′	119

TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; IL-18, interleukin-18.

2.6 Enzyme-linked immunosorbent assay (ELISA)

ELISA kits were used to quantify the amounts of TNF-α, IL-1β, and IL-18 in the cultured HPMVECs’ supernatant (cat. no. ml077385, ml058059, and ml058055; Shanghai Enzyme-linked Biotechnology Co., Ltd, Shanghai, China). A microplate reader served for measuring the absorbance at 450 nm.

2.7 Western blot (WB) analysis

To conduct WB analysis, whole cell extracts were prepared. About 50 μg protein aliquot underwent 10% SDS-PAGE separation, and was moved onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Following 2 h blockage in 5% fat-free milk, blots received one night of culture using primary antibodies at 4°C. The following primary antibodies were used: anti-P2X7R (1:1,000; cat. no. 13809; Cell Signaling Technology, Danvers, MA, USA), anti-NLRP3 (1:1,000; cat. no. ab4207; Abcam), anti-Caspase 1(1:1,000; cat. no. 22915-1-AP; San Ying Biotechnology, Wuhan, China), anti-GSDMD (1:1,000; cat. no. ab210070; Abcam), anti-cleaved N-terminal-GSDMD (N-GSDMD) (1:1,000; cat. no. ab215203; Abcam), anti-GADPH (1:1,000; cat. no. ET1601-4; HuaAn Biotechnology Co., Ltd, Hangzhou, China), and anti-β-actin (1:1,000; cat. no. HA722023; HuaAn Biotechnology Co., Ltd). Following washing, the blots underwent another 2 h of incubation using secondary antibodies (1:1,000; Beyotime Institute of Biotechnology). The membranes were rinsed and targeted bands were identified using an enhanced chemiluminescence system (cat. no. 32109;Pierce, USA;). Labworks Analysis Software (Upland, CA, USA) was used for quantitative analysis of protein densitometry, which was then normalized to the levels of housekeeping proteins GADPH or β-actin.

2.8 Statistical analysis

Experimental data analysis relied on the SPSS 23.0, and data presentation followed mean ± standard deviation format. Statistical distinctions among groups were established through one-way ANOVA, succeeded by the least significant difference t-test. P < 0.05 reported statistical significance.

3 Results

3.1 Characterization of HPMVECs

Cultured HPMVECs were characterized by observing morphology, performing ICC, and using TEM. Phase-contrast light microscopy revealed that HPMVECs retained the classic cobblestone morphology characteristic of endothelial cells (Figure 1a). ICC staining confirmed the presence of endothelial-specific markers CD34 and factor VIII-related antigen in the cells (Figure 1b and c). Additionally, the morphology of Weibel-Palade bodies, specific morphological markers of endothelial cells, was observed by TEM. These rod-shaped organelles were present in cultured HPMVECs (Figure 1d).

Figure 1

Culture and identification of HPMVECs. (a) HPMVEC morphology after 2 days in culture, observed under a light microscope. Scale bar = 100 μm. (b) and (c) ICC for endothelial cell-specific markers CD34 and Factor VIII. Both markers showed positive expression in cultured cells. Scale bar = 100 μm. (d) Ultrastructural morphology of Weibel-Palade bodies (indicated by arrows) in HPMVECs, observed by TEM. Scale bar = 2 μm.

3.2 P2X7R activation in hyperoxia-induced injury in HPMVECs

The impact of P2X7R on hyperoxic injury in HPMVECs was investigated by measuring cell viability with a CCK-8 assay. As depicted in Figure 2a, elevated exposure to hyperoxia was accompanied by obviously weakened cell viability (P < 0.01). To further explore the role of P2X7R during hyperoxia, the receptor was blocked with A438079, a P2X7R antagonist. It was found that cell death induced by hyperoxia was reduced by approximately 20 and 27% after 24 and 48 h, respectively, with 10 μM A438079 incubation, versus the hyperoxia group (P < 0.01). This concentration was used in subsequent analyses of P2X7R function.

Figure 2

Effects of blocking P2X7R on HPMVECs in hyperoxia. (a) Cell viability assessed by CCK-8 assay. A438079, a P2X7R antagonist, was used at concentrations of 5, 10, and 20 μM. Viability was higher in the hyperoxia + A438079 groups than in the hyperoxia-only group. (b) Protein expression levels of P2X7R were determined by WB. GAPDH served as the loading control. Histograms represent the relative quantitative evaluation of protein levels. *P < 0.05 and **P < 0.01 versus the control group; ^# P < 0.05 and ^## P < 0.01 versus the hyperoxia group.

P2X7R expression was then evaluated by WB analysis (Figure 2b). Hyperoxia was found to elevate the expression of P2X7R in HPMVECs (P < 0.01). Treatment with the inhibitor A438079 partially reversed the hyperoxia-induced elevation in P2X7R expression (P < 0.01), implicating that P2X7R participates in hyperoxic injury in HPMVECs.

3.3 Hyperoxia-induced pyroptosis in HPMVECs through P2X7R activation

Pyroptosis was observed in the hyperoxia group (Figure 3a). The role of P2X7R in this process was then explored. P2X7R acts as a critical inflammation switch, mediating key downstream responses that activate inflammasomes, including the NLRP3 inflammasome. The NLRP3 inflammasome activates the protease caspase-1, inducing GSDMD-dependent pyroptosis.

Figure 3

Effects of blocking P2X7R on NLRP3-mediated pyroptosis in hyperoxia. (a) Microscopic images of HPMVECs in control and hyperoxia groups, with arrows indicating cells exhibiting pyroptotic morphology. Scale bar = 20 μm. (b)–(e) Protein expression levels of caspase-1 (b), NLRP3 (c), GSDMD (d), and N-GSDMD (e) were determined by WB. GAPDH served as the loading control. Histograms represent the relative quantitative evaluation of protein levels. *P < 0.05 and **P < 0.01 versus the control group; ^# P < 0.05 and ^## P < 0.01 versus the hyperoxia group.

WB analysis confirmed higher caspase-1, NLRP3, GSDMD, and N-terminal GSDMD levels in the hyperoxia group relative to controls (P < 0.01, Figure 3b–e). After administration of A438079, a decrease in the protein expression of these markers was noted, indicating that blocking P2X7R reduced NLRP3 inflammasome activation (P < 0.01, Figure 3b–e).

Both ELISA and RT-PCR were employed to measure TNF-α, IL-1β, and IL-18 levels, aiming at exploring how pyroptosis affected the secretion of pro-inflammatory cytokines. According to analysis results, the hyperoxia group exhibited remarkably higher TNF-α concentrations versus the control at both mRNA and protein levels (P < 0.01, Figure 4a and d). Lower levels were observed following treatment with A438079 (P < 0.01). A similar pattern was noted for IL-1β and IL-18 (P < 0.01, Figure 4b, c, e, and f). Thus, these data suggest that P2X7R is associated with hyperoxia-induced pyroptosis and the downstream inflammatory response in HPMVECs.

Figure 4

Effects of blocking P2X7R on proinflammatory factors in hyperoxia. (a)–(c) Protein levels of TNF-α, IL-1β, and IL-18, determined by ELISA. (d)–(f) Relative mRNA expression of TNF-α, IL-1β, and IL-18, assessed by RT-qPCR. *P < 0.05 and **P < 0.01 versus the control group; ^# P < 0.05 and ^## P < 0.01 versus the hyperoxia group.

3.4 Inhibition of NLRP3 alleviated HPMVEC injury induced by P2X7R activation

The role of P2X7R was further verified by examining the cell viability of HPMVECs in response to the selective P2X7R agonist BzATP at concentrations of 25 and 50 µM. Dose–response effects showed decreased viability in the BzATP group (P < 0.01, Figure 5). The involvement of the NLRP3 pathway was tested using the NLRP3 antagonist glibenclamide at concentrations of 100 and 200 µM. Results demonstrated that glibenclamide alleviated BzATP-induced cell injury, supporting the NLRP3 pathway’s role downstream of P2X7R activation. Concentrations of 200 µM BzATP and 50 µM glibenclamide were selected for subsequent experiments.

Figure 5

Effects of P2X7R/NLRP3 signaling on cell viability of HPMVECs. Cell viability assessed by CCK-8 assay. Cells were treated with BzATP, a P2X7R agonist, at concentrations of 100 and 200 μM. Glibenclamide, used to inhibit NLRP3 inflammasome activation at concentrations of 25 and 50 μM, ameliorated HPMVEC injury induced by P2X7R activation. *P < 0.05 and **P < 0.01 versus the control group; ^# P < 0.05 and ^## P < 0.01 versus the BzATP group (100 μM). ^▲ P < 0.05 and ^▲▲ P < 0.01 versus the BzATP group (200 μM).

3.5 P2X7R activated the NLRP3 inflammasome to induce pyroptosis in HPMVECs

Finally, we investigated whether P2X7R activation affects HPMVEC viability through NLRP3 activation and pyroptosis. Stimulation of P2X7R with BzATP (200 µM) made pyroptosis-related markers more greatly expressed and promoted TNF-α, IL-1β, and IL-18 to be well secreted (P < 0.01, Figure 6). Conversely, the NLRP3 antagonist glibenclamide (50 µM) effectively reduced these expressions and secretions in HPMVECs (Figure 7). In summary, these results indicate that P2X7R may mediate pyroptosis through the NLRP3 inflammasome.

Figure 6

Effects of P2X7R/NLRP3 signaling on pyroptosis in HPMVECs. Cells were treated with BzATP (200 µM) to activate P2X7R. Glibenclamide (50 µM) was used to inhibit NLRP3 inflammasome activation. (a) Microscopic images of HPMVECs in control and BzATP treatment groups, with arrows indicating pyroptotic cells. Scale bar = 20 μM. (b)–(e) Protein expression levels of caspase-1 (b), NLRP3 (c), GSDMD (d), and N-GSDMD (e) were determined by WB. GAPDH and β-actin served as loading controls. *P < 0.05 and **P < 0.01 versus the control group; ^# P < 0.05 and ^## P < 0.01 versus the BzATP group.

Figure 7

Effects of P2X7R/NLRP3 signaling on proinflammatory factors in HPMVECs. Cells were treated with BzATP (200 µM) to activate P2X7R. Glibenclamide (50 µM) was used to inhibit NLRP3 inflammasome activation. (a)–(c) Protein levels of TNF-α, IL-1β, and IL-18, determined by ELISA. (d)–(f) Relative mRNA expression of TNF-α, IL-1β, and IL-18, assessed by RT-qPCR. *P < 0.05 and **P < 0.01 versus the control group; ^# P < 0.05 and ^## P < 0.01 versus the BzATP group.

4 Discussion

This study probed the impact of P2X7R on hyperoxia-induced damage to HPMVECs in vitro. Findings indicate that hyperoxia notably decreases HPMVEC viability and elevates caspase-1, NLRP3, GSDMD, N-GSDMD, TNF-α, IL-1β, and IL-18 levels. Additionally, blocking P2X7R enhanced cell viability and reduced pyroptosis and inflammation following hyperoxia exposure. Moreover, treatment with glibenclamide to block NLRP3 significantly reduced pyroptosis induced by P2X7R activation. Our findings suggest a correlation between hyperoxic HPMVEC injury and the P2X7R/NLRP3-activated pyroptotic pathway.

Long-term exposure can disrupt normal pulmonary alveolarization and vascularization, increasing the risk of developing BPD. Previous research has indicated that impaired vascularization exacerbates alveolar simplification and contributes to pulmonary hypertension in BPD [20–22]. Therefore, strategies to protect PMVECs from hyperoxic injury may lead to effective therapeutic agents for BPD. In addition, it has been noted that infants with BPD have elevated levels of IL-1β, IL-6, IL-8, IL-10, IL-33, and TNF-α. This emphasizes the crucial role of inflammation in BPD progression [23–25].

P2X7R, a purinergic receptor, when stimulated, facilitates IL-1β and IL-18 to be released. In contrast, P2X7R-knockout mice do not release IL-1β following ATP stimulation [15]. P2X7R shows a strong expression in multiple cell types inside the lungs, including type I alveolar epithelial cells and PMVECs. In a mouse model of hyperoxic acute lung damage, its critical function in inflammatory processes has been confirmed [15,26]. Hyperoxia activates P2X7R, initiating K⁺ efflux and secretion of proinflammatory cytokines [27], suggesting its involvement in hyperoxic injury through the inflammatory response. However, research studies have not well explained its precise contribution to BPD progression. Our research endeavors to delineate the role of P2X7R in hyperoxic injury within PMVECs.

There is evidence to suggest that P2X7R strongly stimulates the NLRP3 inflammasome, resulting in inflammasome assembly and IL-1β and IL-18 release. The synthesis of IFN-γ, which is crucial for inflammatory responses and adaptive immunity, is facilitated by IL-1β, while IL-18 greatly promotes immune cell recruitment to damaged tissues [28]. Consequently, this research investigates whether P2X7R induces hyperoxic injury in HPMVECs via the NLRP3-mediated pyroptotic pathway. Pyroptosis, a caspase-dependent programmed cell death pathway, excels in producing pro-inflammatory factors (IL-1β and IL-18). Overactive pyroptosis can result in significant pathological harm. Numerous studies have underscored the association between pyroptosis and diverse inflammatory conditions, including sepsis, neurodegenerative disorders, inflammatory bowel disease, acute lung injury, non-alcoholic fatty liver disease, atherosclerosis, and cancer [29–34]. When pyroptosis occurs, the NLRP3 inflammasome sets off the canonical inflammasome pathway. The NLRP3 inflammasome, of which the activation could be achieved by exogenous or endogenous factors, as a result, caspase-1 became matured and IL-1β and IL-18 were secreted [12]. The release of N-GSDMD occurs upon the cleavage of GSDMD by activated caspase-1. After transferring to the plasma membrane, this N-GSDMD generates pores and triggers an inflammatory reaction. The conversion of caspase-1 precursors into corresponding active forms is facilitated by N-GSDMD, IL-1β, and IL-18 [35,36].

In this study, blockade of the P2X7R/NLRP3 pathway only partially alleviated hyperoxia-induced injury in HPMVECs, suggesting the involvement of other mechanisms. Various signaling pathways participate in pyroptosis. Gaidt et al. reported that Toll-like receptor (TLR) 4-TRIF-RIPK1-FADD-CASP8 signaling serves as an alternative pathway to activate NLRP3 inflammasome in human monocytes, independent of P2X7R activation [37]. He et al. showed that in murine dendritic cells, prolonged LPS exposure activated NLRP3 inflammasome and promoted IL-1β production via TLR signaling, in a P2X7-independent manner [38]. These findings indicate that NLRP3 is a convergent point for inflammatory cytokine release, some aspects of which may occur independently of P2X7R. Additionally, the NLRP1 inflammasome and NLRC4 inflammasome are also central in the canonical inflammasome pathway of pyroptosis [39,40]. Besides inflammasome-dependent pathways, other signaling mechanisms contribute to pyroptosis. Yersinia infections in macrophages trigger caspase-8 cleavage of GSDMD, which in turn initiates pyroptosis [41], and granzyme A, a lymphocyte-derived enzyme, cleaves GSDMB, which in turn triggers pyroptosis in a number of cancer cell types [42].

Furthermore, the pathways through which hyperoxia causes cellular damage are multifaceted and not entirely elucidated. Hyperoxia promotes endogenous reactive oxygen species (ROS) to be excessively produced, instigating an inflammatory response. Elevated ROS concentrations result in DNA damage, cell membrane lipid peroxidation, and the activation of genes associated with inflammation and cellular mortality [43]. In addition to pyroptosis, apoptosis represents another prevalent cell death mechanism triggered by hyperoxia. Various pathways, including Nrf2, NF-κB, and MAPK, can induce apoptosis upon exposure to hyperoxic conditions [44]. Thus, alleviating endothelial/epithelial cell apoptosis may well benefit the BPD treatment.

5 Conclusion

In summary, activation of P2X7R is implicated in hyperoxic injury of HPMVECs through NLRP3 inflammasome-related pyroptosis. These findings identify P2X7R as a potential therapeutic strategy targeting BPD prevention and treatment. Nevertheless, the P2X7R/NLRP3-mediated pyroptotic pathway is not the sole mechanism governing hyperoxic injury in HPMVECs, indicating the need for future studies to explore additional mechanisms involved.

Funding information: This work was completed with the support from Sichuan Science and Technology Program (grant number 2024NSFSC1669) and Chengdu Medical Research Project (grant number 2022082).
Author contributions: W.Z. took charge of study designing, experiments, data analysis, and manuscript writing. Z.D. performed cell culture, the CCK-8 assay, and ELISA. H.L. performed WB and qRT-PCR. S.G. performed cell culture and ICC. R.J. was responsible for data collection and analysis, and manuscript revision. The published version of the manuscript had been read by all authors and obtained their approval.
Conflict of interest: Authors state no conflict of interest.
Data availability statement: The datasets generated and/or analyzed during the current study can be obtained from the corresponding author on reasonable request.

References

[1] Stoll BJ, Hansen NI, Bell EF, Walsh MC, Carlo WA, Shankaran S, et al. Trends in care practices, morbidity, and mortality of extremely preterm neonates, 1993–2012. JAMA. 2015;314(10):1039–51. 10.1001/jama.2015.10244.Search in Google Scholar PubMed PubMed Central

[2] Jensen EA, Edwards EM, Greenberg LT, Soll RF, Ehret DEY, Horbar JD. Severity of bronchopulmonary dysplasia among very preterm infants in the united states. Pediatrics. 2021;148(1):e2020030007. 10.1542/peds.2020-030007.Search in Google Scholar PubMed PubMed Central

[3] Bancalari E, Jain D. Bronchopulmonary dysplasia: 50 years after the original description. Neonatology. 2019;115(4):384–91. 10.1159/000497422.Search in Google Scholar PubMed

[4] Jobe AJ. The new BPD: an arrest of lung development. Pediatr Res. 1999;46(6):641–3. 10.1203/00006450-199912000-00007.Search in Google Scholar PubMed

[5] De Paepe ME, Mao Q, Powell J, Rubin SE, DeKoninck P, Appel N, et al. Growth of pulmonary microvasculature in ventilated preterm infants. Am J Respir Crit Care Med. 2006;173(2):204–11. 10.1164/rccm.200506-927OC.Search in Google Scholar PubMed PubMed Central

[6] Stevens RP, Paudel SS, Johnson SC, Stevens T, Lee JY. Endothelial metabolism in pulmonary vascular homeostasis and acute respiratory distress syndrome. Am J Physiol Lung Cell Mol Physiol. 2021;321(2):L358–76. 10.1152/ajplung.00131.2021.Search in Google Scholar PubMed PubMed Central

[7] Zhang X, Lu A, Li Z, Sun J, Dai D, Qian L. Exosomes secreted by endothelial progenitor cells improve the bioactivity of pulmonary microvascular endothelial cells exposed to hyperoxia in vitro. Ann Transl Med. 2019;7(12):254. 10.21037/atm.2019.05.10.Search in Google Scholar PubMed PubMed Central

[8] Yee M, White RJ, Awad HA, Bates WA, McGrath-Morrow SA, O’Reilly MA. Neonatal hyperoxia causes pulmonary vascular disease and shortens life span in aging mice. Am J Pathol. 2011;178(6):2601–10. 10.1016/j.ajpath.2011.02.010.Search in Google Scholar PubMed PubMed Central

[9] Hafner C, Wu J, Tiboldi A, Hess M, Mitulovic G, Kaun C, et al. Hyperoxia induces inflammation and cytotoxicity in human adult cardiac myocytes. Shock. 2017;47(4):436–44. 10.1097/SHK.0000000000000740.Search in Google Scholar PubMed

[10] Bustani P, Kotecha S. Role of cytokines in hyperoxia mediated inflammation in the developing lung. Front Biosci. 2003;8:s694–704. 10.2741/1113.Search in Google Scholar PubMed

[11] Kalikkot Thekkeveedu R, Guaman MC, Shivanna B. Bronchopulmonary dysplasia: a review of pathogenesis and pathophysiology. Respir Med. 2017;132:170–7. 10.1016/j.rmed.2017.10.014.Search in Google Scholar PubMed PubMed Central

[12] Kelley N, Jeltema D, Duan Y, He Y. The NLRP3 inflammasome: an overview of mechanisms of activation and regulation. Int J Mol Sci. 2019;20(13):3328. 10.3390/ijms20133328.Search in Google Scholar PubMed PubMed Central

[13] Paik S, Kim JK, Silwal P, Sasakawa C, Jo EK. An update on the regulatory mechanisms of NLRP3 inflammasome activation. Cell Mol Immunol. 2021;18(5):1141–60. 10.1038/s41423-021-00670-3.Search in Google Scholar PubMed PubMed Central

[14] Liu Q, Niu C, Zhang Q, Sun S, Chen Y, Shi Y. Amitriptyline inhibits NLRP3 inflammasome activation via the ASM/CE pathway in a cell model of NAFLD. Biocell. 2024;48(5):759–69. 10.32604/biocell.2024.048551.Search in Google Scholar

[15] Galam L, Rajan A, Failla A, Soundararajan R, Lockey RF, Kolliputi N. Deletion of P2X7 attenuates hyperoxia-induced acute lung injury via inflammasome suppression. Am J Physiol Lung Cell Mol Physiol. 2016;310(6):L572–81. 10.1152/ajplung.00417.2015.Search in Google Scholar PubMed PubMed Central

[16] Kong H, Zhao H, Chen T, Song Y, Cui Y. Targeted P2X7/NLRP3 signaling pathway against inflammation, apoptosis, and pyroptosis of retinal endothelial cells in diabetic retinopathy. Cell Death Dis. 2022;13(4):336. 10.1038/s41419-022-04786-w.Search in Google Scholar PubMed PubMed Central

[17] Martínez-García JJ, Martínez-Banaclocha H, Angosto-Bazarra D, de Torre-Minguela C, Baroja-Mazo A, Alarcón-Vila C, et al. P2X7 receptor induces mitochondrial failure in monocytes and compromises NLRP3 inflammasome activation during sepsis. Nat Commun. 2019;10(1):2711. 10.1038/s41467-019-10626-x.Search in Google Scholar PubMed PubMed Central

[18] McClenahan D, Hillenbrand K, Kapur A, Carlton D, Czuprynski C. Effects of extracellular atp on bovine lung endothelial and epithelial cell monolayer morphologies, apoptoses, and permeabilities. Clin Vaccine Immunol. 2009;16(1):43–8. 10.1128/CVI.00282-08.Search in Google Scholar PubMed PubMed Central

[19] Mekala N, Gheewala N, Rom S, Sriram U, Persidsky Y. Blocking of P2X7R reduces mitochondrial stress induced by alcohol and electronic cigarette exposure in brain microvascular endothelial cells. Antioxidants (Basel). 2022;11(7):1328. 10.3390/antiox11071328.Search in Google Scholar PubMed PubMed Central

[20] Hansmann G, Sallmon H, Roehr CC, Kourembanas S, Austin ED, Koestenberger M, et al. Pulmonary hypertension in bronchopulmonary dysplasia. Pediatr Res. 2021;89(3):446–55. 10.1038/s41390-020-0993-4.Search in Google Scholar PubMed PubMed Central

[21] Durlak W, Thébaud B. The vascular phenotype of BPD: new basic science insights-new precision medicine approaches. Pediatr Res. 2024;96(5):1162–71. 10.1038/s41390-022-02428-7.Search in Google Scholar PubMed

[22] Arjaans S, Wagner BD, Mourani PM, Mandell EW, Poindexter BB, Berger RMF, et al. Early angiogenic proteins associated with high risk for bronchopulmonary dysplasia and pulmonary hypertension in preterm infants. Am J Physiol Lung Cell Mol Physiol. 2020;318(4):L644–54. 10.1152/ajplung.00131.2019.Search in Google Scholar PubMed PubMed Central

[23] Yoon BH, Romero R, Jun JK, Park KH, Park JD, Ghezzi F, et al. Amniotic fluid cytokines (interleukin-6, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-8) and the risk for the development of bronchopulmonary dysplasia. Am J Obstet Gynecol. 1997;177(4):825–30. 10.1016/s0002-9378(97)70276-x.Search in Google Scholar PubMed

[24] Rocha G, Proença E, Guedes A, Carvalho C, Areias A, Ramos JP, et al. Cord blood levels of IL-6, IL-8 and IL-10 may be early predictors of bronchopulmonary dysplasia in preterm newborns small for gestational age. Dis Markers. 2012;33(1):51–60. 10.3233/DMA-2012-0903.Search in Google Scholar PubMed PubMed Central

[25] Cakir U, Tayman C, Yucel C. A novel diagnostic marker for the severity of bronchopulmonary dysplasia in very low birth weight infants: interleukin-33. Pediatr Allergy Immunol Pulmonol. 2019;32(1):12–7. 10.1089/ped.2019.0994.Search in Google Scholar PubMed PubMed Central

[26] Wang S, Zhao J, Wang H, Liang Y, Yang N, Huang Y. Blockage of P2X7 attenuates acute lung injury in mice by inhibiting NLRP3 inflammasome. Int Immunopharmacol. 2015;27(1):38–45. 10.1016/j.intimp.2015.04.035.Search in Google Scholar PubMed PubMed Central

[27] Kolliputi N, Shaik RS, Waxman AB. The inflammasome mediates hyperoxia-induced alveolar cell permeability. J Immunol. 2010;184(10):5819–26. 10.4049/jimmunol.0902766.Search in Google Scholar PubMed PubMed Central

[28] Dinarello CA. Immunological and inflammatory functions of the interleukin-1 family. Annu Rev Immunol. 2009;27:519–50. 10.1146/annurev.immunol.021908.132612.Search in Google Scholar PubMed

[29] Wen R, Liu YP, Tong XX, Zhang TN, Yang N. Molecular mechanisms and functions of pyroptosis in sepsis and sepsis-associated organ dysfunction. Front Cell Infect Microbiol. 2022;12:962139. 10.3389/fcimb.2022.962139.Search in Google Scholar PubMed PubMed Central

[30] Wu KJ, Wang WR, Cheng QH, Li H, Yan WZ, Zhou FR, et al. Pyroptosis in neurodegenerative diseases: from bench to bedside. Cell Biol Toxicol. 2023;39(6):2467–99. 10.1007/s10565-023-09820-x.Search in Google Scholar PubMed

[31] Zheng W, Liu C. E3 ubiquitin ligase CBL-B suppresses vascular endothelial cell pyroptosis and injury in intracranial aneurysm by facilitating NLRP3 degradation. Biocell. 2024;48(2):293–301. 10.32604/biocell.2023.044211.Search in Google Scholar

[32] Svegliati-Baroni G, Pierantonelli I, Torquato P, Marinelli R, Ferreri C, Chatgilialoglu C, et al. Lipidomic biomarkers and mechanisms of lipotoxicity in non-alcoholic fatty liver disease. Free Radic Biol Med. 2019;144:293–309. 10.1016/j.freeradbiomed.2019.05.029.Search in Google Scholar PubMed

[33] Liu B, He R, Zhang L, Hao B, Jiang W, Wang W, et al. Inflammatory caspases drive pyroptosis in acute lung injury. Front Pharmacol. 2021;12:631256. 10.3389/fphar.2021.631256.Search in Google Scholar PubMed PubMed Central

[34] Wang W, Zhang L, Sun Z. Eliciting pyroptosis to fuel cancer immunotherapy: mechanisms and strategies. Cancer Biol Med. 2022;19(7):948–64. 10.20892/j.issn.2095-3941.2022.0049.Search in Google Scholar PubMed PubMed Central

[35] Shi J, Zhao Y, Wang K, Shi X, Wang Y, Huang H, et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature. 2015;526(7575):660–5. 10.1038/nature15514.Search in Google Scholar PubMed

[36] Evavold CL, Ruan J, Tan Y, Xia S, Wu H, Kagan JC. The pore-forming protein gasdermin D regulates interleukin-1 secretion from living macrophages. Immunity. 2018;48(1):35–44.e6. 10.1016/j.immuni.2017.11.013.Search in Google Scholar PubMed PubMed Central

[37] Gaidt MM, Ebert TS, Chauhan D, Schmidt T, Schmid-Burgk JL, Rapino F, et al. Human monocytes engage an alternative inflammasome pathway. Immunity. 2016;44(4):833–46. 10.1016/j.immuni.2016.01.012.Search in Google Scholar PubMed

[38] He Y, Franchi L, Núñez G. TLR agonists stimulate Nlrp3-dependent IL-1β production independently of the purinergic P2X7 receptor in dendritic cells and in vivo. J Immunol. 2013;190(1):334–9. 10.4049/jimmunol.1202737.Search in Google Scholar PubMed PubMed Central

[39] Barnett KC, Li S, Liang K, Ting JP. A 360° view of the inflammasome: mechanisms of activation, cell death, and diseases. Cell. 2023;186(11):2288–312. 10.1016/j.cell.2023.04.025.Search in Google Scholar PubMed PubMed Central

[40] Guo Q, Wu Y, Hou Y, Liu Y, Liu T, Zhang H, et al. Cytokine secretion and pyroptosis of thyroid follicular cells mediated by enhanced NLRP3, NLRP1, NLRC4, and AIM2 inflammasomes are associated with autoimmune thyroiditis. Front Immunol. 2018;9:1197. 10.3389/fimmu.2018.01197.Search in Google Scholar PubMed PubMed Central

[41] Sarhan J, Liu BC, Muendlein HI, Li P, Nilson R, Tang AY, et al. Caspase-8 induces cleavage of gasdermin D to elicit pyroptosis during Yersinia infection. Proc Natl Acad Sci USA. 2018;115(46):E10888–97. 10.1073/pnas.1809548115.Search in Google Scholar PubMed PubMed Central

[42] Zhou Z, He H, Wang K, Shi X, Wang Y, Su Y, et al. Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells. Science. 2020;368(6494):eaaz7548. 10.1126/science.aaz7548.Search in Google Scholar PubMed

[43] Janssen YM, Van Houten B, Borm PJ, Mossman BT. Cell and tissue responses to oxidative damage. Lab Invest. 1993;69(3):261–74.Search in Google Scholar

[44] Alva R, Mirza M, Baiton A, Lazuran L, Samokysh L, Bobinski A, et al. Oxygen toxicity: cellular mechanisms in normobaric hyperoxia. Cell Biol Toxicol. 2023;39(1):111–43. 10.1007/s10565-022-09773-7.Search in Google Scholar PubMed PubMed Central

Received: 2024-04-19

Revised: 2024-10-29

Accepted: 2024-11-04

Published Online: 2024-12-04

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/med-2024-1097

Keywords for this article

hyperoxia; P2X7 receptor; NLRP3 inflammasome; pyroptosis; pulmonary microvascular endothelial cell

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