Bleomycin-induced Acute Lung Injury Research Articles

Chemoreflex sensitization occurs in both male and female rats during recovery from acute lung injury.

Sex-specific patterns in respiratory conditions, such as asthma, COPD, cystic fibrosis, obstructive sleep apnea, and idiopathic pulmonary fibrosis, have been previously documented. Animal models of acute lung injury (ALI) have offered insights into sex differences, with male mice exhibiting distinct lung edema and vascular leakage compared to female mice. Our lab has provided evidence that the chemoreflex is sensitized in male rats during the recovery from bleomycin-induced ALI, but whether sex-based chemoreflex changes occur post-ALI is not known. To bridge this gap, the current study employed the bleomycin-induced ALI animal model to investigate sex-based differences in chemoreflex activation during the recovery from ALI. ALI was induced using a single intra-tracheal instillation of bleomycin (bleo, 2.5mg/Kg) (day 1). Resting respiratory frequency (fR) was measured at 1-2 days pre-bleo, day 7 (D7) post-bleo, and 1 month (1 mth) post-bleo. The chemoreflex responses to hypoxia (10% O2, 0% CO2) and normoxic-hypercapnia (21% O2, 5% CO2) were measured before bleo administration (pre-bleo) and 1 mth post-bleo using whole-body plethysmography. The apnea-hypopnea Index (AHI), post-sigh apneas, and sighs were measured at each time point. There were no significant differences in resting fR between male and female rats at the pre-bleo time point or in the increase in resting fR at D7 post-bleo. At 1 mth post-bleo, the resting fR was partially restored in both sexes but the recovery towards normal ranges of resting fR was significantly lower in male rats. The AHI, post-sigh apneas, and sighs were not different between male and female rats pre-bleo and 1 mth post-bleo. However, at D7 post-bleo, the male rats exhibited a higher AHI than female rats. Both male and female rats exhibited a sensitized chemoreflex in response to hypoxia and normoxic-hypercapnia with no significant differences between sexes. A sex difference in resting ventilatory parameters occurs post ALI with a prolonged increase in resting fR and larger AHI in male rats. On the other hand, we did not find any sex differences in the chemoreflex sensitization that occurs at 1 mth post-bleo. This work contributes to a better understanding of sex-based variations in lung disorders.

Lipocalin-2 as a prognostic marker in patients with acute exacerbation of idiopathic pulmonary fibrosis

BackgroundLipocalin-2 (LCN2) is a secretory glycoprotein upregulated by oxidative stress; moreover, patients with idiopathic pulmonary fibrosis (IPF) have shown increased LCN2 levels in bronchoalveolar lavage fluid (BALF). This study aimed to determine whether circulatory LCN2 could be a systemic biomarker in patients with IPF and to investigate the role of LCN2 in a bleomycin-induced lung injury mouse model.MethodsWe measured serum LCN2 levels in 99 patients with stable IPF, 27 patients with acute exacerbation (AE) of IPF, 51 patients with chronic hypersensitivity pneumonitis, and 67 healthy controls. Further, LCN2 expression in lung tissue was evaluated in a bleomycin-induced lung injury mouse model, and the role of LCN2 was investigated using LCN2-knockout (LCN2 -/-) mice.ResultsSerum levels of LCN2 were significantly higher in patients with AE-IPF than in the other groups. The multivariate Cox proportional hazards model showed that elevated serum LCN2 level was an independent predictor of poor survival in patients with AE-IPF. In the bleomycin-induced lung injury mouse model, a higher dose of bleomycin resulted in higher LCN2 levels and shorter survival. Bleomycin-treated LCN2 -/- mice exhibited increased BALF cell and protein levels as well as hydroxyproline content. Moreover, compared with wild-type mice, LCN2-/- mice showed higher levels of circulatory 8-isoprostane as well as lower Nrf-2, GCLC, and NQO1 expression levels in lung tissue following bleomycin administration.ConclusionsOur findings demonstrate that serum LCN2 might be a potential prognostic marker of AE-IPF. Moreover, LCN2 expression levels may reflect the severity of lung injury, and LCN2 may be a protective factor against bleomycin-induced acute lung injury and oxidative stress.

Sex differences in Acute Lung Injury (ALI)-Induced Respiratory Functional Changes in a Bleomycin Rat Model

Introduction: Acute lung injury (ALI) is a pathology characterized primarily by profound disruption of the epithelial-endothelial interface, leukocyte migration into the alveolar space, leading to disordered gas exchange and consequential respiratory failure. There exist several animal models to study the pathophysiology of ALI. Bleomycin, a cancer therapeutic, has been shown to consistently induce the ALI phenotype in rodent models. We have previously shown that intra-tracheal bleomycin instillation in rats produces deleterious changes in respiratory function in both acute and recovery phases of ALI. In this study, we sought to elucidate the effect of sex bleomycin-induced ALI. Methods: To do this, cohorts of male and female Sprague-Dawley rats were measured using whole-body plethysmography (WBP) where the following respiratory parameters were recorded prior to bleomycin exposure (Week 0): Volume of Oxygen consumed normalized to body weight (VO2/kg), Respiratory rate (RR), Tidal Volume normalized to body weight (TV/kg), Minute Ventilation normalized to body weight (MV/kg), enhanced pause (Penh), pause (PAU), ratio rate of achieving peak expiatory flow (Rpef), peak inspiratory flow (box) (PIFb), peak expiratory flow box (PEFb), inspiratory time (Ti), expiratory time (Te), expiratory flow at the point of 50%TV (EF50), end-inspiratory pause (EIP), end-expiratory pause (EEP), relaxation time (Tr) and time of brake (TB). A single dose of intra-tracheal bleomycin (2.5mg/kg) was instilled and subsequent WBP recordings were performed weekly during the acute phase of ALI (Week 1), and the recovery phase of ALI (Week 4). Results: Our results show that bleomycin-induced ALI resulted in greater DTE (Weeks 1-3) and DTR (Weeks 1-2) in females compared to males. Measures of airway obstruction were also shown to be sex-dependent, as DRpef (Week 1), DPAU (Weeks 2-4), and DPenh (Weeks 2-4) were significantly different between males and females. EF50, a metric highly correlated with lung pathologies, remained unchanged between sexes post-ALI. While inducing ALI did not alter DPIFb between the sexes, DPEFb was elevated among males compared to females (Weeks 2,4). DEIP and DEEP both showed sex-dependent changes post-ALI, with females displaying a greater decrease compared to males. Additionally, there was a greater decrease in DTV/kg among females compared to males post-ALI (Weeks 1-3). DRR, DMV/kg, and DVO2/kg were unchanged across the 4 weeks post-ALI between sexes. Conclusion: Our study reveals sex-specific nuances in bleomycin-induced acute lung injury (ALI) in Sprague-Dawley rats. Differential responses in airway obstruction measures and distinct impacts on respiratory dynamics underscore the importance of considering sex in researching ALI. This work is supported by NIH R01HL152160-01. This is the full abstract presented at the American Physiology Summit 2024 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 Sex-based Differences in Chemoreflex during Recovery from Acute Lung Injury

Introduction- Sex-specific patterns in respiratory conditions, such as asthma, COPD, cystic fibrosis, obstructive sleep apnea, and idiopathic pulmonary fibrosis, have been previously documented. Animal models of acute lung injury (ALI) have offered insights into sex differences, with male mice exhibiting distinct lung edema and vascular leakage compared to female mice. Our lab has provided evidence that the peripheral (or carotid body) chemoreflex is sensitized in male rats during the recovery from bleomycin-induced ALI, but whether sex-based chemoreflex changes occur post-ALI in not known. To bridge this gap, our current study employed the bleomycin-induced ALI animal model to investigate sex-based differences in chemoreflex activation during the recovery of ALI. Methods- ALI was induced using a single intra-tracheal instillation of bleomycin (bleo) (day 1). Resting fR (respiratory frequency) was measured at pre-bleo, day 7 (D7) post-bleo, and 1 month (1 mth) post-bleo time points. The chemoreflex response to hypoxia (10% O2, 0% CO2) and normoxic-hypercapnia (21% O2, 5% CO2) were measured before bleo administration (pre-bleo) time point and 1 month (1 mth) post-bleo using whole-body plethysmography. Apnea-hypopnea Index (AHI), post-sigh apneas, and sighs were measured at each time point. Results- Resting fR: There were no significant differences in resting fR, either at pre-bleo time point or at day 7 (D7) post-bleo between male and female rats. At 1 mth post-bleo, the resting fR was partially restored in both sexes but the recovery towards normal ranges of resting fR was significantly slower in male rats compared to female rats. AHI, post-sigh apneas, and sighs: At the pre-bleo and 1 mth post-bleo time points, there were no significant differences in AHI, post-sigh apneas, and sighs between male and female rats. However, at D7 post-bleo, the male rats exhibited a higher AHI than female rats; Chemoreflex sensitivity: Both male and female rats exhibited a sensitized chemoreflex activation to hypoxia and normoxic-hypercapnia with no significant differences between sexes. Discussion- Our study showed a sex difference in resting ventilatory parameters including resting fR and AHI post ALI. On the other hand, we did not find any sex difference in chemoreflex sensitization at 1 mth post-bleo. This research contributes to a better understanding of sex-based variations in lung disorders. This study was supported by NIH grant R01 HL-152160 and in part, by NIH grants R01 HL-121012 and R01 HL126796. H-JW was also supported by Margaret R. Larson Professorship in Anesthesiology. IZ was partially supported by the Theodore F. Hubbard Foundation. KK was supported by the AHA predoctoral fellowship -AHA award #903872 by the American Heart Association. This is the full abstract presented at the American Physiology Summit 2024 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.

Evaluation of interleukin-1 and interleukin-6 receptor antagonists in a murine model of acute lung injury.

The acute exudative phase of acute respiratory distress syndrome (ARDS), a severe form of respiratory failure, is characterized by alveolar damage, pulmonary oedema, and an exacerbated inflammatory response. There is no effective treatment for this condition, but based on the major contribution of inflammation, anti-inflammatory strategies have been evaluated in animal models and clinical trials, with conflicting results. In COVID-19 ARDS patients, interleukin (IL)-1 and IL-6 receptor antagonists (IL-1Ra and IL-6Ra, kineret and tocilizumab, respectively) have shown some efficacy. Moreover, we have previously developed novel peptides modulating IL-1R and IL-6R activity (rytvela and HSJ633, respectively) while preserving immune vigilance and cytoprotective pathways. We aimed to assess the efficacy of these novel IL-1Ra and IL-6Ra, compared to commercially available drugs (kineret, tocilizumab) during the exudative phase (day 7) of bleomycin-induced acute lung injury (ALI) in mice. Our results first showed that none of the IL-1Ra and IL-6Ra compounds attenuated bleomycin-induced weight loss and venous increase. Histological analyses and lung water content measurements also showed that these drugs did not improve lung injury scores or pulmonary oedema, after the bleomycin challenge. Finally, IL-1Ra and IL-6Ra failed to alleviate the inflammatory status of the mice, as indicated by cytokine levels and alveolar neutrophil infiltration. Altogether, these results indicate a lack of beneficial effects of IL-1R and IL-6R antagonists on key parameters of ALI in the bleomycin mouse model.

Function of KvLQT1 potassium channels in a mouse model of bleomycin-induced acute lung injury.

Acute respiratory distress syndrome (ARDS) is characterized by an exacerbated inflammatory response, severe damage to the alveolar-capillary barrier and a secondary infiltration of protein-rich fluid into the airspaces, ultimately leading to respiratory failure. Resolution of ARDS depends on the ability of the alveolar epithelium to reabsorb lung fluid through active transepithelial ion transport, to control the inflammatory response, and to restore a cohesive and functional epithelium through effective repair processes. Interestingly, several lines of evidence have demonstrated the important role of potassium (K+) channels in the regulation of epithelial repair processes. Furthermore, these channels have previously been shown to be involved in sodium/fluid absorption across alveolar epithelial cells, and we have recently demonstrated the contribution of KvLQT1 channels to the resolution of thiourea-induced pulmonary edema in vivo. The aim of our study was to investigate the role of the KCNQ1 pore-forming subunit of KvLQT1 channels in the outcome of ARDS parameters in a model of acute lung injury (ALI). We used a molecular approach with KvLQT1-KO mice challenged with bleomycin, a well-established ALI model that mimics the key features of the exudative phase of ARDS on day 7. Our data showed that KvLQT1 deletion exacerbated the negative outcome of bleomycin on lung function (resistance, elastance and compliance). An alteration in the profile of infiltrating immune cells was also observed in KvLQT1-KO mice while histological analysis showed less interstitial and/or alveolar inflammatory response induced by bleomycin in KvLQT1-KO mice. Finally, a reduced repair rate of KvLQT1-KO alveolar cells after injury was observed. This work highlights the complex contribution of KvLQT1 in the development and resolution of ARDS parameters in a model of ALI.

Bleomycin-induced modulations of PARP 1 activity, NAD+ and PARG content in rat lung nuclei

Bleomycin-induced lung pathology in rodents is a well recognized animal model widely used for evaluation of new therapeutic approaches in treatment of lung inflammation and fibrotic diseases. It is documented that poly(ADP-ribose)polymerase 1 activity has a significant role in development of inflammatory processes in the heart, liver and brain. Herein, we used biochemical and immunochemical methods for estimation of poly(ADP-ribose)polymerase 1 (PARP 1) activity, NAD+ and poly(ADP-ribose)glycohydrolase (PARG) protein content in rat lung nuclei during the inflammatory phase in a bleomycin-induced one-hit rat model. To evaluate the influence of bleomycin – induced alterations in DNA structure on regulation of poly(ADP-ribose)polymerase 1 activation pathways, we isolated DNA from nuclei of lung tissues in the phase of acute lung inflammation induced by bleomycin, and DNA melting profiles were investigated. In the present study we investigated whether naturally occurring water-soluble polyphenol tannic acid with widely accepted anti-fibrotic and anti-inflammatory effects can influence poly(ADP-ribose)polymerase 1 activity, NAD+ and poly(ADP-ribose)glycohydrolase protein content in nuclear fraction isolated from rat lung tissues in a bleomycin-induced acute lung injury model. It was demonstrated that NAD+ level and poly(ADP-ribose)glycohydrolase protein content decreased in rat lung nuclei during the inflammatory phase in the bleomycin-induced acute lung injury model. Treatment of rats with tannic acid enhanced the effects displayed by bleomycin in lung nuclei, thus indicating synergistic interaction with the drug in the field covering PARP 1 activity, poly(ADP-ribose)glycohydrolase (PARG) protein and NAD+ content in lung nuclei. We observed PARP 1 inhibition in nuclei of lung tissue during the inflammatory phase in the bleomycin-induced acute lung injury rat model, which could be coupled with the drop of NAD+ level in nuclei. In the present study we highlighted that bleomycin (BLM) can induce DNA destabilization in lung nuclei. It was proposed that bleomycin-induced modulations in DNA structure could hamper PARP 1 binding with DNA and down-regulate the enzyme activating pathway in lung nuclei. The role of poly(ADP-ribose)glycohydrolase depletion in lung nuclei and sequential accumulation of poly(ADP-ribose)polymers in lung cells, which triggers their destruction and tissues damage, was proposed. It is suggested that in the light of synergistic interaction between bleomycin and tannic acid (TA) the anti-inflammatory role of tannic acid should be repurposed.

NMDAR activation attenuates the protective effect of BM-MSCs on bleomycin-induced ALI via the COX-2/PGE2 pathway

N-methyl-d-aspartate (NMDA) receptor (NMDAR) activation mediates glutamate (Glu) toxicity and involves bleomycin (BLM)-induced acute lung injury (ALI). We have reported that bone marrow-derived mesenchymal stem cells (BM-MSCs) are NMDAR-regulated target cells, and NMDAR activation inhibits the protective effect of BM-MSCs on BLM-induced pulmonary fibrosis, but its effect on ALI remains unknown. Here, we found that Glu release was significantly elevated in plasma of mice at d 7 after intratracheally injected with BLM. BM-MSCs were pretreated with NMDA (the selective agonist of NMDAR) and transplanted into the recipient mice after the BLM challenge. BM-MSCs administration significantly alleviated the pathological changes, inflammatory response, myeloperoxidase activity, and malondialdehyde content in the damaged lungs, but NMDA-pretreated BM-MSCs did not ameliorate BLM-induced lung injury in vivo. Moreover, NMDA down-regulated prostaglandin E2 (PGE2) secretion and cyclooxygenase (COX)-2 expression instead of COX-1 expression in BM-MSCs in vitro. We also found that NMDAR1 expression was increased and COX-2 expression was decreased, but COX-1 expression was not changed in primary BM-MSCs of BLM-induced ALI mice. Further, the cultured supernatants of lipopolysaccharide (LPS)-pretreated RAW264.7 macrophages were collected to detect inflammatory factors after co-culture with NMDA-pretreated BM-MSCs. The co-culture experiments showed that NMDA precondition inhibited the anti-inflammatory effect of BM-MSCs on LPS-induced macrophage inflammation, and PGE2 could partially alleviate this inhibition. Our findings suggest that NMDAR activation attenuated the protective effect of BM-MSCs on BLM-induced ALI in vivo. NMDAR activation inhibited COX-2 expression and PGE2 secretion in BM-MSCs and weakened the anti-inflammatory effect of BM-MSCs on LPS-induced macrophage inflammation in vitro. In conclusion, NMDAR activation attenuates the protective effect of BM-MSCs on BLM-induced ALI via the COX-2/PGE2 pathway. Keywords: Acute Lung Injury, BM-MSCs, NMDA receptor, COX-1/2, PGE2.

Jingfang Granules (荆防颗粒) alleviates bleomycin-induced acute lung injury through regulating PI3K/Akt/mTOR signaling pathway

Ethnopharmacological relevanceAcute lung injury is a kind of clinical emergency severe syndrome which might trigger acute respiratory distress syndrome. Jingfang Granules (荆防颗粒) is a traditional Chinese medicine which has been proven to improve acute lung injury induced by bleomycin through inhibiting recruitment and overactive of inflammation. However, the potential mechanisms are still not well evaluated. Aim of studyThe aim of this study was to evaluate the protective function of Jingfang Granules on bleomycin caused acute lung injury and further discuss the potential pharmacological mechanisms. Materials and methodsC57BL/6J mice were intratracheal injected bleomycin to induce model with acute lung injury. The protective impact of Jingfang Granules on acute lung injury and lung fibrosis triggered by bleomycin were evaluated through detecting micebody weight, lung appearance, lung index, and histopathology. The potential pharmacological mechanism of Jingfang Granules in treating acute lung injury was further elucidated by the methods of network pharmacology, proteomics, metabolomics, as well as western blot. Additionally, the network pharmacology analysis and molecular docking technology were integrated to investigate the targets of Jingfang Granules improving acute lung injury. ResultsOur results indicated that Jingfang Granules effectively protected mice from acute lung injury induced by bleomycin, which was confirmed by higher body weight, lower pulmonary edema and lung index, and improved pathology and fibrosis of lung tissue compared to model group. Proteomics, western blot, and metabolomics were integrated and the results confirmed that Jingfang Granules regulated the Glycolysis/Gluconogenesis and Pyruvate metabolism through downregulating the PI3K/Akt/mTOR signaling pathway. The network pharmacology analysis and molecular docking technology results showed that the targets of Jingfang Granules for treating acute lung injury were enriched in the PI3K/Akt signaling pathway, which included 7 target proteins such as MAPK1, MAPK3, JAK2, HRAS, EGFR, PIK3R1, and PIK3CA. ConclusionThis study indicates that Jingfang Granules displays a markedly protective effect on acute lung injury caused by bleomycin through downregulating PI3K/Akt/mTOR signaling pathway, which in turn regulates Glycolysis/Gluconogenesis and Pyruvate metabolism.

Jingfang Granule alleviates bleomycin-induced acute lung injury via CD200-CD200R immunoregulatory pathway.

Jingfang granules (JF), one famous traditional Chinese formula in "She Sheng Zhong Miao Fang" written by Shi-Che Zhang during the Ming Dynasty era, has been widely used to prevent epidemic diseases in history and now was recommended for the treatment of coronavirus disease 2019 (COVID-19) in China. However, the roles of JF against acute lung injury and its mechanisms remain unclear. Acute lung injury (ALI) and its progressive acute respiratory distress syndrome (ARDS) are a continuum of lung inflammatory disease with high morbidity and mortality in clinic, especially in COVID-19 patients. The present study aims to investigate the effect of JF on ALI and clarify its underlying mechanisms for clinical application in COVID-19 control. Bleomycin-induced ALI mice were given oral gavage daily for seven days with or without Jingfang granules (2, 4g/kg). The body weight, lung wet/dry weight ratios, lung appearance and tissue histopathology were evaluated. Quantitative real-time PCR, biochemical bronchoalveolar lavage fluids analysis was used to determine the gene expression of proinflammation factor and infiltrated inflammatory cells in lung. Immunofluorescence image and western blot were used to detect the markers of alveolar macrophages (AMs), endothelial cell apoptosis and changes of CD200-CD200R pathway. Firstly, histopathological analysis showed that JF significantly attenuated pulmonary injury and inflammatory response in ALI mice. Then, cytokine detection, inflammatory cells assay, and JNKs and p38 pathway analysis indicated that the recruitment and activation of alveolar macrophages was the main reason to cause ALI and JF could reverse this variation. Next, immunofluorescence staining and TUNEL assay showed that JF upregulated the expression of CD200 and suppressed the apoptosis of alveolar endothelial cells. Finally, double immunofluorescence staining of CD200 and CD11c indicated that the seriously damaged tissue had the lower CD200 while more AMs infiltration, which was confirmed by RT-PCR analysis of CD200/CD200R. Jingfang granules can protect lung from acu te injury and mitigate the recruitment and overactive AMs-induced inflammation via CD200-CD200R immunoregulatory signal axis, which will provide an experimental basis for Jingfang granules clinical applications in COVID-19.

Softness enhanced macrophage-mediated therapy of inhaled apoptotic-cell-inspired nanosystems for acute lung injury

Engineered nanosystems offer a promising strategy for macrophage-targeted therapies for various diseases, and their physicochemical parameters including surface-active ligands, size and shape are widely investigated for improving their therapeutic efficacy. However, little is known about the synergistic effect of elasticity and surface-active ligands. Here, two kinds of anti-inflammatory N-acetylcysteine (NAC)-loaded macrophage-targeting apoptotic-cell-inspired phosphatidylserine (PS)-containing nano-liposomes (PSLipos) were constructed, which had similar size and morphology but different Young’s modulus (E) (H, ~ 100 kPa > Emacrophage vs. L, ~ 2 kPa < Emacrophage). Interestingly, these PSLipos-NAC showed similar drug loading and encapsulation efficiency, and in vitro slow-release behavior of NAC, but modulus-dependent interactions with macrophages. Softer PSLipos-L-NAC could resist macrophage capture, but remarkably prolong their targeting effect period on macrophages via durable binding to macrophage surface, and subsequently more effectively suppress inflammatory response in macrophages and then hasten inflammatory lung epithelial cell wound healing. Especially, pulmonary administration of PSLipos-L-NAC could significantly reduce the inflammatory response of M1-like macrophages in lung tissue and promote lung injury repair in a bleomycin-induced acute lung injury (ALI) mouse model, providing a potential therapeutic approach for ALI. The results strongly suggest that softness may enhance ligand-directed macrophage-mediated therapeutic efficacy of nanosystems, which will shed new light on the design of engineered nanotherapeutics.Graphical abstract

Non-biased RNA-Seq Analysis of Cardiac Tissues post Acute Lung Injury

Patients suffering from acute lung injury (ALI) are at a high risk of developing cardiac arrhythmias. Our recent study demonstrated that bleomycin (Bleo)-induced ALI in rats exhibited significantly more spontaneous premature ventricular contractions (PVCs) from one to three weeks post induction. Although spontaneous PVCs disappeared at 4 weeks post ALI, PVCs were evoked again by electrical stimulation of the decentralized stellate ganglia in Bleo-exposed rats but not in normal rats. In the current study, we aimed to utilize non-biased RNA-Seq analysis to identify potential gene candidates in cardiac tissues, which may be associated with cardiac arrhythmia during the recovery from ALI. We created an ALI rat model using a single tracheal instillation of bleomycin (2.5 mg/kg), with saline as a vehicle (Veh) control. Blood troponin level was measured between Veh- and Bleo-treated rats at Week 1 (W1) post Veh/Bleo. Cardiac tissues were harvested at W4 post Veh/Bleo for RNA-seq analysis and following PCR validation. At W1 post ALI, cardiac troponin level was significantly increased in Bleo-treated rats whereas it was undetectable in Veh-treated (Control) rats. RNA-seq analysis indicated 11 downregulated (AABR07045427, Habp2, AC130862, Per2, Coch, Phactr3, Angptl7, LOC103690108, Mthfd2, Sorcs1, U6) and 13 upregulated (Calhm5, Metazoa_SRP, Tgm1, Pcare, Drd4, Selplg, AABR07027722; Rn5-8s, 5_8S_rRNA) genes in cardiac tissues at 4 weeks post ALI vs Veh. PCR validated that the gene expressions of Habp2, Coch, Phactr3, LOC103690108, Mthfd2 were significantly downregulated and the gene expressions of Pcare, Drd4 were significantly upregulated in the hearts of Bleo-treated rats at W4 post ALI. Our study demonstrated that multiple gene changes in cardiac tissues are associated with cardiac injury and arrhythmogenesis in bleomycin-induced ALI in male rats. Future studies will further determine the cause-effect relationship between these altered genes and cardiac arrhythmia post ALI. This study was supported by NIH grant R01 HL-152160 and in part, by NIH grants R01 HL126796. 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.

Attenuation of Ventilation-Enhanced Epithelial–Mesenchymal Transition through the Phosphoinositide 3-Kinase-γ in a Murine Bleomycin-Induced Acute Lung Injury Model

Mechanical ventilation (MV) used in patients with acute lung injury (ALI) induces lung inflammation and causes fibroblast proliferation and excessive collagen deposition-a process termed epithelial-mesenchymal transition (EMT). Phosphoinositide 3-kinase-γ (PI3K-γ) is crucial in modulating EMT during the reparative phase of ALI; however, the mechanisms regulating the interactions among MV, EMT, and PI3K-γ remain unclear. We hypothesized that MV with or without bleomycin treatment would increase EMT through the PI3K-γ pathway. C57BL/6 mice, either wild-type or PI3K-γ-deficient, were exposed to 6 or 30 mL/kg MV for 5 h after receiving 5 mg/kg AS605240 intraperitoneally 5 days after bleomycin administration. We found that, after bleomycin exposure in wild-type mice, high-tidal-volume MV induced substantial increases in inflammatory cytokine production, oxidative loads, Masson's trichrome staining level, positive staining of α-smooth muscle actin, PI3K-γ expression, and bronchial epithelial apoptosis (p < 0.05). Decreased respiratory function, antioxidants, and staining of the epithelial marker Zonula occludens-1 were also observed (p < 0.05). MV-augmented bleomycin-induced pulmonary fibrogenesis and epithelial apoptosis were attenuated in PI3K-γ-deficient mice, and we found pharmacological inhibition of PI3K-γ activity through AS605240 (p < 0.05). Our data suggest that MV augmented EMT after bleomycin-induced ALI, partially through the PI3K-γ pathway. Therapy targeting PI3K-γ may ameliorate MV-associated EMT.

Characterization of acute lung injury in the bleomycin rat model.

The aim of this study was to describe and characterize the pathophysiological changes occurring during the early inflammatory phase (first 3 days) in the rat bleomycin model of lung injury preceding the development of fibrosis. Further, we wanted to understand the kinetics and factors contributing to bleomycin-induced acute lung injury (ALI) and provide a robust, reliable and reproducible framework of features of ALI readouts to assess effects of therapeutics on bleomycin-induced ALI in rats. We induced ALI in rats with intratracheal (i.t.) installation of bleomycin. The animals were sacrificed on predetermined time points, that is, Day 0, 1, 2, and 3 post the bleomycin challenge. We analyzed bronchoalveolar lavage fluid (BALF) and lung tissue to establish and assess relevant experimental features of ALI. We demonstrated that bleomycin induced key features of experimental ALI including a profound increase in neutrophils in BALF (50-60%), pulmonary edema, and lung pathology on Day 3 after challenge. Furthermore, we showed that TGF-β1, IL-1β, TNF-α, IL-6, CINC-1, TIMP-1, and WISP-1 were induced by studying their kinetic profile during the first 3 days after bleomycin injury consistent with their known role ALI. We also confirmed that detectable fibrogenesis occurs at the earliest on Day 3 after injury based on collagen content, along with changes in the TGF-β/Smad signaling pathway and increased expression of Galectin-3, Vimentin, and Fibronectin in lung homogenate. Our report presents robust features and contributing mediators/factors to the pathology of bleomycin-induced ALI in rats on Day 3. The kinetic data provide insights on the progression of ALI and a detailed understanding of early events before actual fibrosis development. This set of experimental endpoints is very appropriate and invaluable for efficacy testing of potential novel therapeutic treatments (single or combined) in ALI and understanding their mechanism of action.

Gut microbiota modulates bleomycin-induced acute lung injury response in mice.

Airway instillation of bleomycin (BLM) in mice is a widely used, yet challenging, model for acute lung injury (ALI) with high variability in treatment scheme and animal outcomes among investigators. Whether the gut microbiota plays any role in the outcome of BLM-induced lung injury is currently unknown. Intratracheal instillation of BLM into C57BL/6 mice was performed. Fecal microbiomes were analyzed by 16s rRNA amplicon and metagenomic sequencing. Germ-free mice conventionalization and fecal microbiota transfer between SPF mice were performed to determine dominant commensal species that are associated with more severe BLM response. Further, lungs and gut draining lymph nodes of the mice were analyzed by flow cytometry to define immunophenotypes associated with the BLM-sensitive microbiome. Mice from two SPF barrier facilities at the University of Chicago exhibited significantly different mortality and weight loss during BLM-induced lung injury. Conventionalizing germ-free mice with SPF microbiota from two different housing facilities recapitulated the respective donors' response to BLM. Fecal microbiota transfer from the facility where the mice had worse mortality into the mice in the facility with more survival rendered recipient mice more susceptible to BLM-induced weight loss in a dominant negative manner. BLM-sensitive phenotype was associated with the presence of Helicobacter and Desulfovibrio in the gut, decreased Th17-neutrophil axis during steady state, and augmented lung neutrophil accumulation during the acute phase of the injury response. The composition of gut microbiota has significant impact on BLM-induced wasting and death suggesting a role of the lung-gut axis in lung injury.

Mitoquinone alleviates bleomycin-induced acute lung injury via inhibiting mitochondrial ROS-dependent pulmonary epithelial ferroptosis.

Numerous studies demonstrated that bleomycin (BLM) caused acute lung injury (ALI). This study explored the role of mitochondrial reactive oxygen species (ROS) on BLM-induced ALI and pulmonary epithelial ferroptosis. Male C57BL/6J mice were intratracheally injected with BLM (3.0mg/kg). BEAS-2B cells, human bronchial epithelial cells, were cultured with BLM (10μg/ml). Pulmonary MDA and 4-HNE, two markers of lipid peroxidation, were elevated in BLM-exposed mice. Oxidized lipids were upregulated in BLM-exposed BEAS-2B cells. Ferroptosis-characteristic ultrastructure, mainly disappearance of mitochondrial bilayer membrane structure and cristae, was observed in BLM-exposed pulmonary epithelium. Ferrostatin-1, a specific inhibitor of ferroptosis, attenuated BLM-evoked pulmonary lipid peroxidation, ferroptosis-characteristic mitochondrial ultrastructure and pulmonary epithelial death. The in vitro experiments showed that mitochondrial membrane potentials (MMPs) were decreased and mitochondrial ROS were increased in BLM-exposed BEAS-2B cells. Mitoquinone (MitoQ), a mitochondria-targeted antioxidant, prevented BLM-induced MMP reduction and mitochondrial ROS elevation in BEAS-2B cells. The in vivo experiment found that MitoQ attenuated BLM-evoked GSH depletion and lipid peroxidation in mouse lungs. Moreover, MitoQ prevented BLM-induced ferroptosis-characteristic mitochondrial changes, pulmonary epithelial death and ALI. In conclusion, mitochondrial ROS are an initiator of BLM-induced pulmonary epithelial ferroptosis. Mitochondria-targeted antioxidants may be used as potential therapeutic agents for BLM-induced ALI.

CNP-miR146a Mitigates Methicillin-Resistant Staphylococcus aureus–Induced Acute Lung Injury by Decreasing Inflammation

INTRODUCTION: ARDS has a nearly 40% in-hospital mortality, and current treatment is largely limited to supportive care. Pneumonia and sepsis are the underlying causes responsible for ARDS in a majority of cases with inflammation as a central pathophysiologic feature. We have previously shown that CNP-miR146a, a radical scavenging cerium oxide nanoparticle (CNP) conjugated to the anti-inflammatory microRNA (miR)-146a, reduces bleomycin-induced acute lung injury by decreasing inflammation and oxidative stress. We therefore hypothesized that CNP-miR146a would decrease inflammation in methicillin-resistant Staphylococcus aureus (MRSA)–induced acute lung injury. METHODS: C57BL/6 mice (n = 46) were given IT MRSA or saline (control) followed 4 hours later by 1 ng/50 μL IT CNP-miR146a (MRSA + CNP-miR146a) or saline (control, MRSA). At 24 hours after infection, bronchoalveolar lavage fluid was analyzed by ELISA for proinflammatory cytokines. Whole lungs were processed for total RNA extraction, and quantitative polymerase chain reaction analysis was performed to evaluate proinflammatory gene expression. Statistical significance was determined by 1-way ANOVA (p < 0.05 significant). RESULTS: MRSA-injured lungs had significantly higher relative gene expression of proinflammatory IL-8 (p = 0.0056), IL-1β (p = 0.0023), and TNFα (p = 0.0024) compared with controls. Treatment with CNP-miR146a significantly lowered the expression of IL-6 (p = 0.0271), IL-8 (p = 0.0232), IL-1β (p = 0.0499), and TNFα (p = 0.0121). MRSA injury also significantly increased bronchoalveolar lavage fluid IL-6 protein concentration (p = 0.0011) compared with control, and treatment with CNP-miR146a returned this to control levels (p = 0.0227; Figure).Figure.: CNP-miR146a, a radical scavenging cerium oxide nanoparticle conjugated to the anti-inflammatory microRNA-146a; MRSA, methicillin-resistant Staphylococcus aureus.CONCLUSION: CNP-miR146a rescues the MRSA-injured lung by decreasing inflammation and bronchoalveolar proinflammatory protein leak, and therefore has significant promise as a potential therapeutic for ARDS.

Histopathological, physiological and biochemical assessment of resveratrol nanocapsules efficacy in bleomycin-induced acute and chronic lung injury in rats

Acute lung injury (ALI) is a life-threatening illness which may progress to chronic pulmonary fibrosis (CPF). Resveratrol (RSV), a natural polyphenol, is known to exert several pharmacological effects on lung injury. However, its physicochemical properties and pharmacokinetic profile limit its clinical applications. In this study, RSV was loaded into lipid nanocapsules (LNCs) aiming to overcome these limitations. RSV-LNCs were prepared by phase inversion method and showed small uniform particle size (∼55 nm, PdI 0.04) with high entrapment efficiency >99%. The efficacy of RSV-LNCs in the prophylaxis against ALI and treatment of CPF was investigated in bleomycin-induced lung injury. For assessment of ALI, rats were administered a single oral dose of RSV (10 mg/kg) either free or as RSV-LNCs 4 h before bleomycin and euthanized 3 days later. For CPF, treatments in the same dose were given daily from days 10–20 after bleomycin and rats were euthanized on day-21. Results showed enhanced beneficial role for RSV-LNCs, compared to RSV, in the prevention of ALI as demonstrated by preservation of pulmonary microscopic and ultrastructural architecture and improvement of pulmonary functions. Analysis of BALF revealed reduction in oxidative stress markers, IL-6 level, leukocytosis and neutrophilia. iNOS and c-caspase 3 immunohistochemical expression and CD68+ cells immunofluorescence were inhibited. However, RSV-LNCs failed to show any improvement in oxidative stress, chronic inflammation, apoptosis and collagen deposition in CPF. In conclusion, RSV-LNCs are promising nanoplatforms for mitigating ALI detrimental effects. Future research investigating higher doses and longer durations of treatment is recommended to evaluate RSV-LNCs anti-fibrotic potential in CPF.

Pulmonary Delivery of Hydroxychloroquine Nanostructured Lipid Carrier as a Potential Treatment of COVID-19.

Coronavirus Disease 2019 (COVID-19) is a pandemic caused by severe acute respiratory syndrome coronavirus 2. Pneumonia is considered the most severe and long-term complication of COVID-19. Among other drugs, hydroxychloroquine (HCQ) was repurposed for the management of COVID-19; however, low efficacy and cardiac toxicity of the conventional dosage form limited its use in COVID-19. Therefore, utilizing nanotechnology, a pulmonary delivery system of HCQ was investigated to overcome these limitations. HCQ was formulated in nanostructured lipid carriers (HCQ-NLCs) using the hot emulsification–ultrasonication method. Furthermore, the prepared formulation was evaluated in vitro. Moreover, the efficacy was tested in vivo in a bleomycin-induced acute lung injury mice model. Intriguingly, nanoformulations were given by the intratracheal route for 6 days. HCQ-NLCs showed a mean particle size of 277 nm and a good drug release profile. Remarkably, acute lung injury induced by bleomycin was associated with a marked elevation of inflammatory markers and histological alterations in lung tissues. Astoundingly, all these changes were significantly attenuated with HCQ-NLCs. The pulmonary delivery of HCQ-NLCs likely provided adequate targeting to lung tissues. Nevertheless, there is hope that this novel strategy will eventually lead to the improved effectiveness and diminished probability of alarming adverse drug reactions.

Influenza A Virus Causes Shedding of the Alveolar Epithelial Glycocalyx through Activation of Sheddases

ObjectiveThe healthy alveolar epithelium is protected by a heparan sulfate rich, glycosaminoglycan layer called the epithelial glycocalyx. Our group found that the epithelial glycocalyx is shed in patients with acute respiratory distress syndrome (ARDS). In murine models of LPS‐ or bleomycin‐induced acute lung injury, sheddases (membrane‐bound enzymes that cleave extracellular potions of transmembrane proteins) are upregulated and associated with glycocalyx shedding and increased lung permeability. ARDS is commonly caused by viral infections including influenza A (IAV). In murine models, IAV causes massive and persistent glycocalyx shedding into the airspace but the mechanisms by which this occurs are unknown. The objective of this work is to determine the molecular processes underlying IAV‐induced shedding of the glycocalyx.HypothesisWe hypothesize that IAV causes glycocalyx shedding through induction of host sheddases.MethodsWe examined the literature and curated a list of sheddases associated with IAV with potential to cleave the glycocalyx (MMP‐7, ‐2, ‐9 and their inhibitors TIMP‐1 and ‐2). C57BL/6 mice were infected intranasally with A/PR/8/34 (H1N1) at 30,000 PFU/mouse and bronchoalveolar lavage and lung tissue were collected at day 1, 3, and 7 post infection. Sheddase expression was assessed by RT‐qPCR and RNAscope was used to localize lung sheddase expression in infected and uninfected lungs. MLE‐12 mouse lung epithelial cells were infected with viable or heat‐inactivated (56C for 30 min) A/PR/8/34 (H1N1) at a MOI of 1 and sheddase expression measured by RT‐qPCR.ResultsMice infected with IAV develop significant lung inflammation (increased BAL inflammatory cells), lung permeability (increased BAL protein), and increased glycocalyx shedding. MMP‐7 is upregulated in infected vs. uninfected lungs at day 1 and 3 post infection, then returns to baseline levels by day 7. MMP‐7 is only expressed in cells that are directly infected by IAV. Expression of the MMP‐7 inhibitor TIMP‐1 is similar to uninfected lungs on day 1, but increases 50‐fold on day 3. In contrast, MMP‐2 and MMP‐9, as well as their inhibitor TIMP‐2 are not upregulated in the first 7 days after IAV infection. Preliminary studies in lung epithelial cells suggest that heat‐inactivated IAV fails to upregulate MMP‐7.ConclusionsTogether, these data suggest that localized IAV infection increases MMP‐7 in a murine model of IAV infection, but has no effect on several other sheddases. This suggests that MMP‐7 may modulate IAV‐induced glycocalyx shedding. Future studies will explore the mechanisms of IAV induced glycocalyx shedding which could provide molecular targets for clinical intervention in IAV‐ARDS pathogenesis.