Background Oxygen therapy in preterm neonates is associated with airway hyperreactivity. The role of Rho/Rho‐kinase smooth muscle signaling in hyperoxia‐induced airway hyperreactivity remains understudied. We hypothesized that inhibition of Rho‐kinase will attenuate airway hyperreactivity induced by neonatal hyperoxia. Methods Newborn rats were raised in hyperoxia (>95% O2) or ambient air (AA) for 7 days. Subgroups were injected with a Rho‐kinase inhibitor: Y‐27632 (10 mg·kg−1·day−1) or fasudil (10 mg·kg−1·day−1), or a FP receptor antagonist ‐ AS604872 (30 mg·kg−1·day−1). After exposures, tracheal cylinders were prepared for in vitro wire myography. Contraction to methacholine or PGF2α was measured in the presence or absence of tissue‐bath Y‐27632, fasudil, or AS604872. Lung PGF2α levels, Rho‐kinase protein level and Rho‐kinase 1 activity were measured by ELISA. Results Tracheal smooth muscle contraction was significantly greater in hyperoxic compared to AA groups. Both, Y‐27632 and fasudil significantly decreased contractility to MCh or PGF2α in hyperoxic groups versus hyperoxic controls (p < 0.001), but did not alter AA group responses. Inhibition of FP receptors attenuated responses to PGF2α. Hyperoxia significantly increased lung PGF2α compared to AA (p < 0.01), but Rho‐kinase inhibition did not influence PGF2α level. Rho‐kinase protein level (p < 0.001) and activity (p < 0.01), were increased by hyperoxia, but blockade of FP receptor reduced the Rho‐kinase 1 activity (p < 0.05) under hyperoxic condition. Conclusions This study demonstrates an active role of Rho/Rho‐kinase signaling on hyperoxia‐induced airway hyperreactivity. These findings suggest that Rho‐kinase inhibitors might serve as an effective therapy for hyperoxia‐induced airway hyperreactivity.

Background This study was undertaken to test the hypothesis that the newly synthesized curcuminoids B2BrBC and C66 supplementation will overcome hyperoxia‐induced tracheal hyperreactivity and impairment of relaxation of tracheal smooth muscle (TSM). Materials and methods Rat pups (P5) were exposed to hyperoxia (>95% O2) or normoxia for 7 days. At P12, tracheal cylinders were used to study in vitro contractile responses induced by methacholine (10−8–10−4M) or relaxation induced by electrical field stimulation (5–60 V) in the presence/absence of B2BrBC or C66, or to study the direct relaxant effects elicited by both analogs. Results Hyperoxia significantly increased contraction and decreased relaxation of TSM compared to normoxia controls. Presence of B2BrBC or C66 normalized both contractile and relaxant responses altered by hyperoxia. Both, curcuminoids directly induced dose‐dependent relaxation of preconstricted TSM. Supplementation of hyperoxic animals with B2BrBC or C66, significantly increased catalase activity. Lung TNF‐α was significantly increased in hyperoxia‐exposed animals. Both curcumin analogs attenuated increases in TNF‐α in hyperoxic animals. Conclusion We show that B2BrBC and C66 provide protection against adverse contractility and relaxant effect of hyperoxia on TSM, and whole lung inflammation. Both analogs induced direct relaxation of TSM. Through restoration of catalase activity in hyperoxia, we speculate that analogs are protective against hyperoxia‐induced tracheal hyperreactivity by augmenting H2O2 catabolism. Neonatal hyperoxia induces increased tracheal contractility, attenuates tracheal relaxation, diminishes lung antioxidant capacity, and increases lung inflammation, while monocarbonyl CUR analogs were protective of these adverse effects of hyperoxia. Analogs may be promising new therapies for neonatal hyperoxic airway and lung disease.

Neonatal hyperoxia induces airway hyperreactivity by potentiating contractile responses and impairing the relaxation of airway smooth muscle (ASM). Maintenance of balance between these processes in airways is critical for normal flow of air, and non-adrenergic- noncholinergic inhibitory system is an important component of airway smooth muscle tone. Therefore, we tested the hypothesis that exogenous pituitary adenylate cyclase activating peptide (PACAP) will restore the impaired relaxant responses of tracheal smooth muscle exposed to neonatal hyperoxia. Tracheal cylinders were obtained from Wistar rat pups (P10) exposed to hyperoxia (≥ 95% O2) or room air for seven days. These cylinders were used to study relaxant responses of ASM evoked by electrical field relaxation (EFS) in precontracted tissues (carbachol, 5x10μM) in absence or presence of a single dose of PACAP (50 nM). In addition, the dose-response relaxant effect of PACAP (1nM – 1μM) was tested in absence or presence of a PAC1 receptor antagonist; a protein kinase A (PKA) inhibitor (Rp-8-CPT-cAMPS); a phosphodiesterase 4 (PDE4) inhibitor (rolipram); or a large-conductance calcium-activated potassium channel blocker (charybdotoxin). The relaxation is expressed as percentage of preconstricted state, and data are presented as mean±s.e.m. Hyperoxic exposure significantly decreased the relaxant responses of ASM towards EFS, as compared to those obtained from control animals exposed to room air, and these reduced relaxant responses in hyperoxic tissues were significantly reversed in presence of PACAP (p<0.001). PACAP induced dose-dependent relaxation and the relaxant responses were overcompensated in hyperoxic animals. PAC1 antagonist; PKA; PDE4 inhibition; or blockade of calcium-activated potassium channel significantly reduced PACAP-induced relaxation (p<0.01).The results of this study revealed that PACAP reverses the impaired relaxation of airway smooth muscle induced by hyperoxic exposure via cAMP/PKA signaling pathway and through calcium-activated potassium channel. We speculate that use of PACAP might be an effective therapeutic approach to prevent the airway hyperreactivity induced by hyperoxia. Supported by the Ministry of Education, Science, and Technology of the Republic of Kosovo - Small Research Grants: 2-2459-11 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The use of oxygen therapy (high doses of oxygen - hyperoxia) in the treatment of premature infants results in their survival. However, it also results in a high incidence of chronic lung disease known as bronchopulmonary dysplasia, a disease in which airway hyper-responsiveness and pulmonary hypertension are well known as consequences. In our previous studies, we have shown that hyperoxia causes airway hyper-reactivity, characterized by an increased constrictive and impaired airway smooth muscle relaxation due to a reduced release of relaxant molecules such as nitric oxide, measured under in vivo and in vitro conditions (extra- and intrapulmonary) airways. In addition, the relaxation pathway of the vasoactive intestinal peptide (VIP) and/or pituitary adenylate cyclase activating peptide (PACAP) is another part of this system that plays an important role in the airway caliber. Peptide, which activates VIP cyclase and pituitary adenylate cyclase, has prolonged airway smooth muscle activity. It has long been known that VIP inhibits airway smooth muscle cell proliferation in a mouse model of asthma, but there is no data about its role in the regulation of airway and tracheal smooth muscle contractility during hyperoxic exposure of preterm newborns.

Prolonged oxygen exposure of preterm infants results in the development of bronchopulmonary dysplasia (BPD), a chronic lung disease characterized with lung inflammation, remodeling and airway reactivity. Unfortunately, therapeutic options are limited. Resveratrol is known to have anti‐inflammatory and anti‐oxidant properties in different diseases, however it is not clear about its effects on BPD. In this study we investigated the effects of resveratrol in a rat pup model of BPD. We hypothesize that supplementation of rat pups with resveratrol prevents the lung inflammation induced by hyperoxia. At P5 rat pups were assigned either to room air (21% O2)‐ or hyperoxic (≥95% O2)‐groups and exposed for seven days. During this time, animals were supplemented daily with resveratrol (30 mg/kg of body weight; i.p.) or vehicle. At day P12 animals were euthanized and lungs were harvested, and processed for biochemical and immunohistochemistry studies. Pro‐inflammatory cytokines’ levels (IL‐1β and TNF‐α) were measured in whole lung homogenate using ELISA method. Immunostaining for T‐lymphocyte marker (CD3), B‐lymphocyte marker (CD20) and CD45 was performed on 5 μm lung sections. Levels of IL‐1β and TNF‐α in lung tissue of hyperoxic group of animals were increased significantly (p < 0.01) compared to room air group animals. Supplementation of hyperoxic animals prevented this increase of IL‐1β and TNF‐α, while did not change in room air animals. In hyperoxic animals there was an abundance of CD3, CD20 and CD45 stained cells in the lungs of rat pups exposed to hyperoxia compared to room air exposed pups. Supplementation of hyperoxic animals with resveratrol reduced the number of CD3, CD20 and CD45 stained cells. We conclude that resveratrol prevents lung inflammation induced by neonatal hyperoxia and we speculate that resveratrol might be an effective therapeutic approach to prevent the adverse effects of neonatal hyperoxia. Support or Funding Information Supported by MEST