---
title: "Connexin 43 in Hyperoxia-Induced Bronchopulmonary Dysplasia and Associated Pulmonary Hypertension"
id: "plos-one-19-role-of-connexin-43-in-hyperoxia-induced-bronchopulmonary-dysplasia-and"
canonical_url: "https://medichelpline.com/clinical-feed/plos-one-19-role-of-connexin-43-in-hyperoxia-induced-bronchopulmonary-dysplasia-and"
content_type: "clinical_feed_article"
specialty: "Pulmonology"
source_name: "PLOS ONE (Medicine)"
source_url: "https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0356364"
published_at: "2026-08-24T14:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Connexin 43 in Hyperoxia-Induced Bronchopulmonary Dysplasia and Associated Pulmonary Hypertension
## Provenance & Clinical Metadata
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- **Specialty:** [Pulmonology](https://medichelpline.com/clinical-feed/pulmonology.md)
- **Primary Source:** PLOS ONE (Medicine)
- **Source URL:** [Original Journal Publication](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0356364)
- **Published At:** 2026-08-24T14:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- Premature infants often require supplemental oxygen, which can cause hyperoxia-driven lung injury and lead to **bronchopulmonary dysplasia (BPD)** with arrested alveolarization and inflammation. - **Connexin 43 (Cx43)** forms gap junctions that regulate intercellular communication in lung cells, including type I/II alveolar epithelial cells, endothelium, macrophages and smooth muscle cells. - Prior models show Cx43 upregulation with lung inflammation and that genetic or pharmacologic reduction of Cx43 can alter inflammatory cell recruitment and airway responses. - This study tested whether pharmacological inhibition of Cx43 with the peptide **43Gap26** modifies hyperoxia-induced experimental BPD and associated **pulmonary hypertension (PH-BPD)** using neonatal rats exposed to 90% O2 for 14 days and human fetal pulmonary artery smooth muscle cells (HfPA-SMC) exposed to 60% O2. - Outcomes assessed included lung morphology and alveolarization, lung function, surfactant protein expression, extracellular matrix markers, macrophage phenotype and cytokine secretion, oxidative stress markers, pulmonary artery remodeling and survival. - In vivo, 43Gap26 reduced hyperoxia-induced **Cx43** overexpression and produced a partial improvement in alveolar structure but did not prevent impaired lung function, decreased surfactant protein-B, extracellular matrix remodeling, M2 macrophage polarization, increased TIMP-1 secretion, pulmonary hypertension, or increased mortality. - In vitro, hyperoxia increased Cx43 expression in HfPA-SMC but 43Gap26 did not change hyperoxia-induced secretion of pro-inflammatory cytokines under the tested conditions. - Classical markers of oxidative damage were not significantly increased in the experimental settings, although heme oxygenase-1 expression was elevated. - The authors conclude that **Cx43-GJ signaling** contributes to regulation of alveolar structure during hyperoxic injury, but selective pharmacological inhibition with 43Gap26 alone is insufficient to prevent the complex structural and vascular pathology characteristic of experimental BPD and PH-BPD. - Methods and additional experimental details are provided in the paper's supporting information; specific quantitative data, statistical values and some experimental details are reported in the full article.
## Clinical Analysis & Structured Key Points
Role of connexin 43 in hyperoxia-induced bronchopulmonary dysplasia and associated pulmonary hypertension | PLOS One Browse Subject Areas ? Click through the PLOS taxonomy to find articles in your field. For more information about PLOS Subject Areas, click here . Article Authors Metrics Comments Media Coverage Reader Comments Figures Figures Abstract Premature infants frequently require oxygen supplementation due to lung immaturity, exposing them to the risk of bronchopulmonary dysplasia (BPD), a chronic lung disease characterized by arrested alveolar growth and inflammation. Connexin 43-dependent gap junctions (Cx43-GJ) regulate intercellular communication during lung development and inflammatory responses, but their involvement in BPD remains incompletely defined. This study aimed to assess the contribution of Cx43-GJ in experimental BPD and to evaluate whether selective pharmacological inhibition of Cx43 modulates hyperoxia-induced lung injury and pulmonary hypertension. Newborn rats were exposed to normoxia or hyperoxia (90% O 2 ) for 14 days and treated daily with the Cx43-GJ inhibitor 43 Gap26 or vehicle. Human fetal pulmonary artery smooth muscle cells (HfPA-SMC) were exposed to 21% O 2 or 60% O 2 and treated with or without 43 Gap26 for 48 hours. Lung morphology, function, surfactant protein expression, extracellular matrix markers, macrophage phenotype, cytokine secretion, and oxidative stress markers were assessed. In vivo , 43 Gap26 reduced hyperoxia-induced Cx43 overexpression and partially improved alveolarization. However, 43 Gap26 failed to prevent alterations in lung function, decreased surfactant protein-B expression, extracellular matrix remodeling, macrophage M2 polarization, increased tissue inhibitor of metalloproteinase-1 secretion, pulmonary hypertension, or mortality. Inhibition of Cx43 was also associated with reduced markers of type II alveolar epithelial cell expression. In HfPA-SMC, 43 Gap26 did not modify hyperoxia-induced secretion of pro-inflammatory cytokines, despite increased Cx43 expression. Classical markers of oxidative damage were not significantly increased under our experimental conditions, although heme oxygenase-1 expression was elevated. Overall, pharmacological inhibition of Cx43 partially restored alveolar structure but did not prevent major pathological features of experimental BPD or pulmonary hypertension. These findings suggest that Cx43-GJ signaling is involved in the regulation of alveolar structure during hyperoxic lung injury, but its pharmacological inhibition alone is insufficient to prevent the complex structural and vascular alterations characteristic of experimental BPD. Citation: Pilard C-M, Gassiat L, Cardouat G, Gauthereau I, Robillard P, Dumas-de-la-Roque E, et al. (2026) Role of connexin 43 in hyperoxia-induced bronchopulmonary dysplasia and associated pulmonary hypertension. PLoS One 21(8): e0356364. https://doi.org/10.1371/journal.pone.0356364 Editor: Christopher Torrens, Royal College of Surgeons in Ireland, IRELAND Received: November 6, 2025; Accepted: July 30, 2026; Published: August 24, 2026 Copyright: © 2026 Pilard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting information files. Funding: CMP was funded by a Medical Research Foundation grant (FDM202006011412). https://www.frm.org/fr?_rsc=1qjz4&gad_source=1&gad_campaignid=22460864545&gclid=Cj0KCQjw8p7GBhCjARIsAEhghZ2TrGWbtM9W8iEd7odYOWT0zf9lBQaXJYVN7kEARLChhUp-N2opPwEaAtXwEALw_wcB. FRM did not play any role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Introduction Bronchopulmonary dysplasia (BPD) was first described in 1967 by Northway et al . [ 1 ], but it continues to be one of the most common complications of prematurity, despite major advances in neonatology over the last couple of decades. Indeed, improved medical management of extreme prematurity (before 28 weeks of amenorrhea) increases survival rates but also the incidence of BPD, which varies between 20–75% depending on the cohort [ 2 ]. BPD is a clinical consequence of prenatal (intra-uterine growth restriction, smoking, chorioamnionitis) and post-natal (oxygen supplementation, mechanical ventilation, sepsis) lung injuries. Indeed, preterm infants are born during the second phase of lung development, which includes the canalicular, saccular and alveolar stages. During these stages, the lung is not yet efficient at gas exchange. Therefore, assisted ventilation and supplemental oxygen will initially support ventilation after birth, but will secondarily lead to severe intrapulmonary inflammation characterized by (i) an increase in pulmonary macrophage infiltration, (ii) an increase in pro-inflammatory cytokines secretion such as interleukin-6 (IL-6) and (iii) an imbalanced M1/M2 macrophage polarization [ 3 – 5 ]. These pro-inflammatory features hurdle to normal pulmonary growth and repair, and are responsible for blunted alveolarization. Abnormal microvascular development also occurs, including pulmonary artery (PA) wall thickening and vascular growth arrest responsible for pulmonary microvasculature rarefaction, leading to pulmonary hypertension associated with BPD (PH-BPD) [ 6 , 7 ]. PH-BPD is a severe complication of BPD affecting nearly 25% of extreme premature infants with moderate to severe BPD [ 8 ] and leading to a mortality rate up to 50% within 2 years of diagnosis [ 6 , 8 , 9 ]. Anti-inflammatory approaches attenuate impaired alveolarization induced by hyperoxia [ 5 ]. However, the functional contribution of inflammatory cells to arrested alveolarization and disturbed vascular development remains largely unclear. Moreover, nowadays, our understanding of normal and aberrant lung development is still poor, and there is an urgent need to discover new pathways involved in alveolar development that could reveal new therapeutic targets to prevent abnormal lung development or promote lung regeneration and repair. Connexin 43 (Cx43), a gap junction (GJ) protein involved in cell-to-cell communication seems to be a new potential key player implicated both in late lung development and in the regulation of the inflammatory response. Indeed, Cx43 is expressed in the mouse embryo from the day of gestation E14.5 and in the adult type I and II alveolar epithelial cells (ATI and II, respectively), and also in pulmonary endothelium, lung macrophages and smooth muscle cells [ 10 ]. Cx43 knockout newborn mice have hypoplastic lungs with narrow airspaces and thicker interalveolar septae, and die rapidly after birth from severe respiratory failure [ 11 ]. In another inflammatory lung disease model of acute respiratory distress syndrome induced by intratracheal instillation of Pseudomonas aeruginosa lipopolysaccharide (LPS), Cx43 knockdown mice (Cx43 +/- mice) have decreased neutrophils in the bronchoalveolar lavage (BAL) fluid compared to wild-type mice, suggesting that Cx43 may play a role in pro-inflammatory cells recruitment into the lung [ 12 ]. In the same way, Cx43 is known to be upregulated in a model of ovalbumin-induced allergic asthma, and its inhibition with the blocking peptide 43 Gap26 reduced airway hyper-responsiveness and eosinophil infiltration [ 13 ]. At last, Qing et al . showed that hyperoxia increased Cx43 expression, apoptosis and reactive oxygen species (ROS) production in lungs from a neonatal model of BPD induced by hyperoxia exposure, and they further demonstrated that inhibition of Cx43 with 43 Gap26 reversed these changes, thus improving alveolarization [ 14 ]. Regarding the role of Cx43 in pulmonary hypertension (PH), a recent study from our laboratory showed that Cx43 expression was increased in pulmonary artery (PA) from patients with chronic-hypoxia-induced PH (CH-PH), and Cx43 +/- mice exposed to chronic hypoxia were partially protected against CH-PH with decreased PA remodeling and inflammatory cell infiltration [ 15 ]. However, the role of Cx43 in BPD and PH-BPD pathogenesis remains unclear. In this work, we hypothesized that Cx43 plays a key role in the arrested alveolarization and disturbed vascular development associated with BPD and PH-BPD, by regulating macrophage-driven inflammatory response. To test our hypothesis, we used a well-known neonatal murine model of BPD induced by chronic exposure to hyperoxia (90% O 2 ) for 14 days, with rat pups treated or not with a selective Cx43 inhibitor, 43 Gap26. Some experiments were also performed in human fetal pulmonary artery smooth muscle cells (HfPA-SMC) exposed to hyperoxia (60% O 2 ) and treated or not with 43 Gap26 for 2 days, in order to examine some mechanistic underpinnings of Cx43 on inflammation and oxidative stress. Methods Detailed methods are available in supporting information. Study design We employed a cross-sectional design combining in vivo investigations in a well-established neonatal rat model of hyperoxia with in vitro analyses using a novel culture system of human fetal pulmonary artery smooth muscle cells (HfPA-SMC) exposed to hyperoxic conditions ( S1 Fig ). The study first examined the effects of the connexin 43 (Cx43) inhibitory peptide 43 Gap26 (GenScript Biotech, Rijswijk, NL) on hallmark features of bronchopulmonary dysplasia (BPD)—including alveolar development, lung function, extracellular matrix (ECM) composition, pulmonary inflammation, and oxidative stress—as well as on parameters of pulmonary hypertension (PH) such as pulmonary artery (PA) pressure, right ventricular (RV) hypertrophy, vascular remodeling, reactivity, and density. Selected in vivo findings were then validated in vitro by assessing Cx43 expression, inflammatory response, and oxidative stress in HfPA-SMC. PH development was confirmed through in vivo hemodynamic assessment by echocardiography and cardiac catheterization. RV hypertrophy was quantified by separating the right ventricle (RV) from the left ventricle (LV) and septum and calculating the Fulton index (RV/[LV + septum] weight ratio). Pulmonary function was measured using the forced oscillation technique. Lung morphometry (alveolarization), vascular remodeling and density, and macrophage infiltration were evaluated by histological staining. Inflammatory markers were quantified by ELISA in both experimental models, while PA reactivity was assessed by myography. Oxidative stress was analyzed by Western blotting. All primary antibodies used for Western blotting and immunostaining experiments are provided in S1 Table . All animal experiments complied with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) and were approved by the local ethics committee (CEEA 50) and French regulatory authorities (protocol no. APAFIS#28597). Animals were randomly assigned to experimental groups, and data from replicate cohorts were pooled. Unless otherwise stated in figure legends, each experiment included at least five animals. The establishment of a human fetal tissue bank (cardiac and pulmonary) and all related procedures were approved by the French Biomedicine Agency (authorization no. PFS15−004, effective since May 13, 2015, with no expiration). Written informed consent was obtained from each donor prior to elective pregnancy termination. Ethical approval for this study was granted by the Research Ethics Committee of Bordeaux University Hospital, and all procedures adhered to the principles of the Declaration of Helsinki. In vitro experiments were conducted using cells from at least five independent donors. Statistical analysis Data are presented as individual values and means ± SEM. The sample size (n) represents the number of independent biological replicates—animals, donors, or HfPA-SMC measurements—as specified in figure legends. Data normality was assessed using the Shapiro–Wilk test. Concentration–response relationships were compared using two-way ANOVA. For comparisons among more than two groups, one-way ANOVA or Kruskal–Wallis tests were applied depending on data distribution, followed by appropriate post hoc analyses. Differences between two groups were evaluated using Student’s t-test. Survival curves were analyzed using the Log-rank test. Statistical analyses were performed using GraphPad Prism (version 9.0.0, GraphPad Software, San Diego, CA, USA). A p-value < 0.05 was considered statistically significant. Results Effect of hyperoxia and Cx43 inhibition on Cx43 expression in both models We firstly showed that hyperoxia induced an increase in Cx43 expression in vivo in rat pups whole lungs (p = 0.0014) but not in their isolated intrapulmonary arteries ( Fig 1A and 1B , respectively). Furthermore, in whole lungs from animals exposed to hyperoxia and treated with 43 Gap26 (Hx Gap), Cx43 inhibition was associated with a significant decrease in Cx43 expression as compared to the hyperoxic group (Hx Ct – p = 0.0353) ( Fig 1A ). Download: PNG larger image TIFF original image Fig 1. Effect of hyperoxia and Cx43 inhibition on Cx43 expression in animal and human models. (A) Cx43 protein expression was analyzed by Western blot in whole lung homogenates from neonatal rats exposed to normoxia (Nx Ct) or hyperoxia (Hx Ct), and daily treated or not with the Cx43 inhibitor 43 Gap26 (Nx Gap or Hx Gap) for 14 days. Representative immunoblots are shown (n = 5–13 per group). Protein levels were normalized to total protein using stain-free (SF) technology and expressed as a percentage of Nx Ct. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, one-way ANOVA followed by Tukey’s post hoc test. (B) Cx43 expression in intrapulmonary arteries from neonatal rats exposed to normoxia (Nx Ct) or hyperoxia (Hx Ct). Representative immunoblots are shown (n = 15–16 per group). Protein levels were normalized to total protein (SF technology) and expressed as a percentage of Nx Ct. Data are presented as mean ± SEM. Unpaired t-test. (C) Cx43 biotinylated membrane fractions obtained from human fetal pulmonary artery smooth muscle cells (HfPA-SMCs) exposed to room air (RA Ct – 21% O 2 ) or hyperoxia (Hx Ct). Cell-surface proteins were labeled using a membrane-impermeable biotin reagent at 4 °C to restrict labeling to extracellularly exposed proteins, followed by streptavidin pull-down. Neg Ctrl (–Biotin) corresponds to samples processed in parallel without biotin labeling and was included to assess non-specific binding to streptavidin beads. Representative immunoblots are shown (left side) (n = 9 donors per group). Protein levels were normalized to total protein (SF technology) and expressed as a percentage of RA Ct. Data are presented as mean ± SEM. * P < 0.05, paired t-test. (D) Total Cx43 protein expression in HfPA-SMC exposed to RA (RA Ct) or hyperoxia (Hx Ct). Representative immunoblots are shown (right side) (n = 9 donors per group). Protein levels were normalized to total protein (SF technology) and expressed as a percentage of RA Ct. Data are presented as mean ± SEM. Paired t-test. https://doi.org/10.1371/journal.pone.0356364.g001 In vitro in HfPA-SMC exposed to hyperoxia, we observed an increase in membrane Cx43 expression (p = 0.0466) ( Fig 1C ) while total Cx43 protein expression remained unchanged ( Fig 1D ). Effect of Cx43 inhibition on alveolarization and lung function in the BPD animal model We then assessed the effects of Cx43 inhibition by 43 Gap26 on the main characteristics of BPD. Regarding alveolarization, we demonstrated that 43 Gap26 treatment significantly prevented hyperoxia-induced hypoalveolarization (p = 0.0465) ( Fig 2A and 2B ). Download: PNG larger image TIFF original image Fig 2. Effect of Cx43 inhibition on alveolarization and lung function in a rat model of bronchopulmonary dysplasia. (A) Mean linear intercept (MLI) measured at postnatal day 14 in neonatal rats exposed to normoxia (Nx Ct) or hyperoxia (Hx Ct), and daily treated or not with the Cx43 inhibitor 43 Gap26 (Nx Gap or Hx Gap). n = 6 rats per group. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01; one-way ANOVA followed by Tukey’s post hoc test. (B) Representative hematoxylin and eosin-stained lung sections from the same experimental groups as in (A). Images shown are representative of n = 6 animals per group. (C–F) Lung function parameters measured in intubated neonatal rats from the same experimental groups as in (A). Parameters include inspiratory capacity (IC, C), respiratory system compliance (Crs, D), tissue damping G (E), and tissue elastance H (F). n = 4–12 animals per group. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; one-way ANOVA followed by Tukey’s post hoc test (C, E, F) or Dunn’s multiple comparisons test (D). https://doi.org/10.1371/journal.pone.0356364.g002 However, when studying pulmonary function by forced oscillation maneuver in vivo , hyperoxia exposure significantly decreased the inspiratory capacity (IC) and Crs (p < 0.001) ( Fig 2C and 2D ), and increased tissue damping G (p = 0.0049) (reflecting increased air resistance in the alveoli), and tissue elastance H (p = 0.0382) (reflecting an increased stiffness of the lung tissue) ( Fig 2E and 2F ). 43 Gap26 treatment did not improve hyperoxia-induced IC, Crs and tissue damping G alteration ( Fig 2C , 2D and 2E ) whereas it worsened tissue elastance H in hyperoxic condition (p = 0.0382) ( Fig 2F ). Moreover, 43 Gap26 treatment in animals maintained in normoxia (Nx Gap) significantly altered IC (p = 0.0032) and tissue damping G (p = 0.0197) compared to the normoxic control group ( Fig 2C and 2E ). Effect of Cx43 inhibition by 43 Gap26 on the expression of both alveolar epithelial cells (ATI and II) markers and the surfactant protein B (SP-B) in the BPD animal model Then, we focused on ATI and ATII cells and we showed that neither hyperoxia nor 43 Gap26 had any effect on ATI cell marker expression ( Fig 3A ). Regarding ATII cell marker expression, we demonstrated that hyperoxia significantly increased ATII cell marker expression (p < 0.0001) ( Fig 3B ), leading to a significant reduction in the ATI (RT1–40) to ATII (pro-SPC) cell marker ratio (p = 0.0141) ( Fig 3C ). Moreover, 43 Gap26 prevented the hyperoxia-induced increase in ATII cell marker expression as compared to the hyperoxic control group (p = 0.006) ( Fig 3B ). Download: PNG larger image TIFF original image Fig 3. Effect of Cx43 inhibition on alveolar epithelial cell markers and surfactant protein B expression in a rat model of bronchopulmonary dysplasia. (A) Expression of RT1-40 glycoprotein (podoplanin), a specific marker of alveolar type I (ATI) epithelial cells, analyzed by Western blot in whole lung homogenates from neonatal rats exposed to normoxia (Nx Ct) or hyperoxia (Hx Ct), and daily treated or not with the Cx43 inhibitor 43 Gap26 (Nx Gap or Hx Gap). Representative immunoblots are shown (n = 5–9 per group). Protein levels were normalized to total protein using stain-free (SF) technology and expressed as a percentage of Nx Ct. Data are presented as mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test. (B) Expression of pro-surfactant protein C (pro-SP-C), a specific marker of alveolar type II (ATII) epithelial cells, assessed by Western blot in whole lungs from the same experimental groups as in (A). Representative immunoblots are shown (n = 5–9 per group). Protein levels were normalized to total protein (SF technology) and expressed as a percentage of Nx Ct. Data are presented as mean ± SEM. *** P < 0.001, **** P < 0.0001; one-way ANOVA followed by Tukey’s post hoc test. (C) ATI/ATII marker expression ratio calculated from data in (A) and (B). This ratio reflects relative protein a
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