Preterm newborns frequently require supplemental oxygen because of immature lungs, and prolonged oxygen exposure is a key contributor to bronchopulmonary dysplasia (BPD), a condition marked by arrested alveolar development, inflammation and disturbed vascular growth. Pulmonary hypertension associated with BPD (PH-BPD) is a serious complication that increases morbidity and mortality in affected infants.
Connexin 43 (Cx43) is a gap junction protein expressed in developing and adult lung cells—including type I and II alveolar epithelial cells, endothelium, macrophages and smooth muscle cells—and has been implicated in lung morphogenesis and inflammatory responses. Prior experimental work indicates that Cx43 expression increases in inflammatory lung disease and that Cx43 modulation can influence leukocyte recruitment, airway reactivity and alveolarization in some models. This study tested whether selective pharmacological inhibition of Cx43 with the mimetic peptide 43Gap26 modifies the structural, inflammatory and vascular consequences of neonatal hyperoxia in an established experimental model of BPD and in cultured human fetal pulmonary artery smooth muscle cells (HfPA-SMC).
The investigators used a neonatal rat model in which newborn pups were exposed to either normoxia or hyperoxia (90% O2) for 14 days to induce features of experimental BPD and associated pulmonary vascular disease. Parallel in vitro experiments exposed HfPA-SMC to either 21% O2 (normoxia) or 60% O2 (hyperoxia) for 48 hours to probe cellular responses relevant to pulmonary vascular pathology.
Detailed methods, including dosing schedules, sample sizes, and specific assays, are provided in the paper's supporting information.
Animals in the hyperoxia model received daily administration of the Cx43-gap-junction inhibitor 43Gap26 or vehicle. In vitro, HfPA-SMC cultures were treated with or without 43Gap26 during hyperoxic exposure. The study examined the effects of Cx43 inhibition on expression of Cx43 itself and on downstream structural, inflammatory and oxidative readouts.
Across in vivo and in vitro experiments the authors evaluated:
Hyperoxia exposure increased pulmonary Cx43 expression. Daily treatment with 43Gap26 reduced hyperoxia-induced Cx43 overexpression and produced a partial restoration of alveolar architecture, indicating some preservation of alveolarization. Despite partial structural improvement, 43Gap26 did not prevent alterations in lung function under hyperoxic conditions. Treatment with the inhibitor also failed to improve survival; mortality remained increased with hyperoxia and was not averted by 43Gap26.
Hyperoxia caused reductions in surfactant protein-B expression and induced extracellular matrix remodeling. Pharmacological inhibition of Cx43 with 43Gap26 did not prevent the decrease in surfactant protein-B nor did it block extracellular matrix changes. The hyperoxia-exposed lungs displayed macrophage phenotype shifts toward an M2-polarized profile and increased secretion of tissue inhibitor of metalloproteinase-1 (TIMP-1); these inflammatory and remodeling-related alterations were not prevented by 43Gap26. Notably, inhibition of Cx43 was associated with reduced markers of type II alveolar epithelial cell expression, suggesting complex effects of blockade on epithelial cell phenotype.
Hyperoxia elicited pulmonary vascular changes consistent with pulmonary hypertension, including pulmonary artery remodeling. Administration of 43Gap26 did not prevent the development of pulmonary hypertension or the vascular structural changes induced by hyperoxia in this model.
In cultured human fetal pulmonary artery smooth muscle cells (HfPA-SMC), exposure to hyperoxia increased Cx43 expression. However, treatment with 43Gap26 did not alter hyperoxia-induced secretion of pro-inflammatory cytokines in these cells under the experimental conditions used.
Classical markers of oxidative damage were not significantly increased in the experimental settings reported, although expression of heme oxygenase-1 was elevated with hyperoxia. The study therefore observed limited evidence for widespread oxidative damage by the assays employed, while antioxidant-response elements such as heme oxygenase-1 were upregulated.
The data indicate that Cx43-GJ signaling participates in regulation of alveolar structure during hyperoxic lung injury: pharmacological inhibition with 43Gap26 reduced Cx43 overexpression and produced a partial improvement in alveolar architecture. However, blockade of Cx43 alone did not prevent major pathological features of experimental BPD, including impaired lung function, surfactant loss, extracellular matrix remodeling, macrophage M2 polarization, increased TIMP-1 secretion, pulmonary hypertension, or mortality.
These findings suggest that while Cx43 contributes to hyperoxia-driven structural changes, selective pharmacological inhibition of Cx43 is insufficient as a standalone therapy to halt the multifactorial processes driving BPD and PH-BPD. The paper notes that some mechanistic details and comprehensive methodological parameters are reported in the supporting information. Limitations inherent to the experimental models and to single-target pharmacologic inhibition are implied by the partial and selective effects observed.
Overall, the study supports a role for Cx43 in alveolar development and hyperoxic lung injury but underscores the complexity of BPD pathogenesis and the need for multimodal strategies to prevent or treat experimental BPD and associated pulmonary hypertension.